Effects of explants and growth regulators in garlic callus formation and plant regeneration

We intended to evaluate the effects of different explants and growth regulators on callus induction and plant regeneration in garlic (Allium sativum L.). Furthermore, we intended to differentiate among different morphological types of callus by light microscopy and to relate them with their abilities to regenerate plants in the red-garlic cultivar 069. A factorial design with BDS—basal Dunstan and Short (1977)—medium, as a control and supplemented with 0.042, 0.42 and 4.24 μM picloram or with 0.045, 0.45 and 4.5 μM 2,4-D, in both cases with and without 4.43 μM N⁶-benzylaminopurine (BAP), was used. The cultures were grown in darkness at 25 ± 2°C and they were subcultured over a 6-month period. Basal plates and meristems were highly responsive explants, while immature umbels and root-tips were less responsive ones, as indicated by percentage of induced callus, growing callus and regenerating callus. The best response was 41% regenerating callus with 0.045 μM 2,4-D and BAP from basal plates while 57, 56 and 20% regenerating callus were obtained with 0.45 μM 2,4-D from meristems, root-tips and immature umbels, respectively. Also, these treatments showed a higher percentage of nodular and embryogenic callus (type I). Thus, it can be concluded that the use of meristems and 2,4-D will enhance callus production and quality, increase plant regeneration and allows to develop a protocol suitable for further transformation experiments in garlic.     

Effect of different plant growth regulators on micro-tuber induction and plant regeneration of Pinellia ternate (Thunb) Briet

An efficient micropropagation system for Pinellia ternate (Thunb) Briet, a traditional Chinese medicinal plant, has been developed. Petiole and lamina of P. ternate were used as explants and cultured on Murashige and Skoog (MS) medium containing different concentrations of different plant growth regulators. The results indicated that low concentration of 2,4-dicholorophenoxy acetic acid (2,4-D), indole-3-acetic acid (IAA) and α-naphthalene acetic acid (NAA) were suitable for micro-tuber induction, but callus induction rate increased with increasing concentrations of growth regulators. Tubers induction rates of petiole and leaf were (81.8 %–100 %) and (89.4 %–96.0 %) respectively, when 0.2 mg l⁻¹ 2, 4-dicholorophenoxy acetic acid, indole-3-acetic acid or α-naphthalene acetic acid were present in the medium. Tubers induction rates of petiole and leaf cultured on MS medium supplemented with 0.2–0.5 mg l⁻¹ 6-benzyl amino purine (6-BAP) were (94.1 %–100 %) and (96.0 %–100 %) respectively. When the concentration of 2,4-dicholorophenoxy acetic acid, α-naphthalene acetic acid and 6-benzyl amino purine was increased to 2.0 mg l⁻¹, callus induction rates of petiole and leaf were 100 % and 98.2 %, 91.0 % and 36.0 %, 62.3 % and 70.0 %, respectively. Different concentration of kinetin (KT) and zeatin (ZT) had no significant effect on micro-tuber induction of petiole. Most petioles showed polarity during the cultivation of explants, when supplemented with different concentrations of auxin or cytokinin in the MS medium.     

Effect of seaweed extracts and plant growth regulators on high-frequency in vitro mass propagation of Lycopersicon esculentum L (tomato) through double cotyledonary nodal explant

An efficient and reproducible two-step in vitro propagation system for tomato (Lycopersicon esculentum) was developed by using the combinations of seaweed biostimulant (Gracilaria edulis and Sargassum wightii) extracts and plant growth regulators (PGRs). Double cotyledonary nodal (DCN) explants of Co-3 cultivar were initially cultured on Murashige and Skoog (MS) and Gamborg’s medium (B5) containing thidiazuron (TDZ) and 6-benzylaminopurine (BA); the best responding cytokinin was tested in combinations with different auxins (NAA, IAA and IBA), and seaweed extracts (G. edulis and S. wightii) of about basal MS medium +10–70% was used for shoot proliferation. The best organogenic culture response was obtained on MS medium fortified with 1.5 mg L^?1 TDZ and 1.5 mg L^?1 IBA. Up to 24 shoots per explants were formed at an optimal duration of exposure to 35 days. Mini shoots of about 3–4 cm were transferred to medium supplemented with MS + iP, MS + zeatin, MS + G. edulis and MS + S. wightii at different concentrations. High frequency of shoot elongation was observed in the medium supplemented with 30% G. edulis (15.2 cm), and profuse rooting was observed in the medium supplemented with 50% S. wightii of about 16.1 cm. Shoot elongation and rooting were observed in the medium supplemented with seaweed extracts. The plantlets were transferred to the plant growth chamber (70% of relative humidity and 9 light cycles) and maintained in it for a week, and then they were transferred to a greenhouse condition. The plant growth chamber to green house transferred plantlets showed an increase in the survival rate from 70 to 85%. Thus a two-step regeneration protocol was developed in this study with a combination of seaweed extracts and PGRs, which provides a basis for the production of transgenics with high frequency and survivability of tomato plants.     

Effect of plant growth regulators on evolution of ethylene and methane by different explants of chickpea

Shoot tips, cotyledonary nodes and hypocotyls of chickpea (Cicer arietinum L.) were grown on 3 media: plant induction medium (PIM), callus induction medium (CIM), and shoot induction medium (SIM). Maximum growth and differentiation was seen in PIM, whereas minimum was observed in CIM. Shoot tips which differentiated to multiple shoots evolved negligible amounts of ethylene. Maximum ethylene evolution was recorded by hypocotyls in PIM. Ethylene appears to have stimulatory effect on shoot bud differentiation in cotyledonary nodes. But in hypocotyls, increased ethylene inhibited growth and differentiation. Calli on media containing only auxin (PIM) evolved significantly more ethylene, whereas those on media with cytokinin (SIM) evolved more methane. Callus forming explants like cotyledonary nodes and hypocotyls evolve more ethylene than shoot tips. This revised version was published online in August 2006 with corrections to the Cover Date.     

Micropropagation of Salvia brachyodon through nodal explants

A protocol for in vitro propagation of Balkan endemic plant Salvia brachyodon Vandas from nodal segments was developed. 6-benzylaminopurine was more effective in axillary buds promotion when compared to thidiazuron. The rooting of regenerated shoots was induced by transferring them to the media supplemented with auxins. All tested auxins (indole-3-acetic acid, indole-3-butyric acid, and α-naphthaleneacetic acid) stimulated the rooting of S. brachyodon shoots. The acclimatization of in vitro rooted shoots was successful.     

A study on the effect of genotypes,plant growth regulators and sugars in promoting plant regeneration via organogenesis from soybean cotyledonary nodal callus

We have developed an efficient protocol for callus induction and plant regeneration in three elite soybean cultivars (Williams 82, Loda and Newton). The technique is most novel in that the shoot buds developed from the nodal callus. Callus induction and subsequent shoot bud differentiation were achieved from the proximal end of cotyledonary explants on modified Murashige and Skoog (MS) media containing 2.26 M 2,4-dichlorophenoxy-acetic acid (2,4-D) and 8.8 M benzyladenine (BAP), respectively. Varying the carbon source optimized the regeneration system further. Among the various carbon sources tested, sorbitol was found to be the best for callus induction and maltose for plant regeneration.     

Rapid in vitro multiplication and plant regeneration from nodal explants of Andrographis paniculata: a valuable medicinal plant

A rapid and efficient method for the large-scale propagation of a highly valuable medicinal plant, Andrographis paniculata Nees, through in vitro culture of nodal explants obtained from 15-d-old aseptic seedling has been developed. High frequency direct shoot proliferation was induced in nodal explants cultured on Murashige and Skoog’s medium supplemented with 6-benzylaminopurine (BAP). Amongst the various cytokinins tested (BAP, kinetin, thidiazuron and 2-isopentyl adenine), BAP proved to be the most effective. The shoot forming capacity of the nodal explants was influenced by the BAP concentration (1–12.5 μM), and the optimal response was observed at 10 μM BAP, which induced an average of 34 shoots in 94% of the cultures within 4 wk. Significant differences were recorded in terms of average number of shoots per explant (8.6–34.1) among the different concentrations of BAP investigated. Concentrations of all cytokinins tested reach a level that can be considered above the optimum level, as marked by a reduced frequency of shoot proliferation. The multiple shoots obtained on various concentrations of BAP failed to elongate even after transfer to hormone-free MS medium. Elongation of the induced shoots was achieved on MS basal medium supplemented with 1.0 μM GA₃ within 2 wk. A proliferating shoot culture was established by repeatedly subculturing the original nodal explants on shoot multiplication medium after each harvest of the newly formed shoots. The explants retained their morphogenic potential even after three harvests. Therefore, in 90 d, about 60–70 shoots were obtained from a single nodal explant and the nodal explants from primary shoots further regenerated equivalent number of shoots, depicting their high frequency regeneration potential in A. paniculata. Rooting was best induced in 94% of shoots cultured on MS medium supplemented with 2.5 μM indole-3-butyric acid (IBA), within a wk. The plantlets were successfully transferred to soil after hardening with a 92% survival rate. The system is rapid: the initiation of shoot buds to the transplanting of regenerants to soil is completed in 8–9 wk.     

Effect of various growth regulators on shoot regeneration of sugarcane

Summary Sugarcane (Saccharum spp. hybrid cv. CP 84-1198) embryogenic calluses were induced from young leaves cultured on modified Murashige and Skoog basal medium supplemented with 13.6 μM 2,4-dichlorophenoxyacetic acid. Five concentrations, 0.5, 1.0, 2.5, 5.0, and 10.0 μM, of five different growth regulators, 6-benzylaminopurine, kinetin, 6-γ,γ-(dimethylallylamino)purine, zeatin, and thidiazuron, were tested with or without 22.5 μM α-naphthaleneacetic acid to compare their ability to induce regeneration from embryogenic callus. After 4 wk on medium, the percentage of shoot meristem induction was evaluated, and after 10 wk the total number of shoots produced, as well as the percentage of shoots greater than 1 cm in length, was obtained. Although it had the lowest percentage of elongated shoots, medium containing thidiazuron alone performed better than all other growth regulators tested, with the highest percentage of shoot induction and the largest number of shoots, particularly at a concentration of 2.5 μM.     

Efficient plant regeneration protocol through callus for Saussurea obvallata (DC.) Edgew. (Asteraceae): effect of explant type, age and plant growth regulators

A callus induction and in vitro plantlet regeneration system for the endangered state flower of Uttaranchal (Saussurea obvallata) was optimized by studying the influence of explant type (root, hypocotyl, cotyledon and leaf), age and different concentrations of plant growth regulators. Explants from 10 to 15-day-old seedlings showed maximum callus induction. Callus formation and shoot differentiation was initiated on Murashige-Skoog (MS) medium containing 6-benzyladenine (BA) and -naphthalene acetic acid (NAA) in all explant types. The best results were obtained using leaf explants: 100% callusing was achieved in MS medium supplemented with 2.5 M BA and 1.0 M NAA, and 100% differentiation along with a multiplication rate of 12 shoots per explant with a combination of 5.0 M BA and 1.0 M NAA. However, the results reflected the existence of high inter-explant variability in response to growth regulators. In vitro rooting of shoots was achieved at an efficiency of 100% in one-half strength MS medium supplemented with 2.5 M indole-3-butyric acid. Application of this protocol has potential for mass multiplication of the target species in a limited time period.     

不同生长调节剂对马蹄金愈伤组织诱导的影响

马蹄金是一种优良的地被兼观赏草坪植物。采用正交设计试验法 ,研究了四种不同生长调节剂对马蹄金子叶、叶片、叶柄和下胚轴愈伤组织诱导的影响。结果表明 :生长调节剂是诱导愈伤组织的关键 ,2 ,4 D对愈伤组织诱导具有显著的影响 ,适宜于马蹄金愈伤组织诱导的培养基及生长调节剂为MS +1 .0mg/L 2 ,4 D +0 .2mg/L 6 BA +0 .2mg/LKT +1 .0mg/Lα NAA。     

In vitro rapid regeneration of plantlets from nodal explants of Mucuna pruriens– a valuable medicinal plant

A rapid and efficient protocol for the large‐scale propagation of a potential medicinal plant, Mucuna pruriens, through in vitro culture of nodal segment explants obtained from 15‐day‐old aseptic seedlings is described. Of the three different cytokinins, 6‐benzyladenine (BA), kinetin (Kin) and 2‐isopentenyl adenine (2‐iP) evaluated as supplements to Murashige and Skoog (MS) medium, BA at an optimal concentration of 5.0 μM was effective in inducing multiple shoots. Strength of the basal media also influenced the efficiency of shoot regeneration. The frequency of shoot regeneration tended to increase when the salt concentration in the basal media was reduced. Highest number of multiple shoots (23.3) and maximum average length (5.6 cm) were standardised on half‐strength MS medium supplemented with 5.0 μM BA along with 0.5 μM α‐naphthalene acetic acid (NAA) at pH 5.8. Rooting was best induced in shoots excised from proliferated shoot cultures on MS medium augmented with an optimal concentration of 1.0 μM indole‐3‐butyric acid (IBA). The in vitro‐raised plantlets with well‐developed shoots and roots were successfully established in earthen pots containing garden soil and were grown in greenhouse with 90% survival rate. The results of this study provide the first report on in vitro plant regeneration of M. pruriens.     

In vitro regeneration from bulb explants of Indian squill,Urginea indica Kunth

Callus cultures were established from bulb explants of diploid Urginea indica Kunth (Indian squill) on a modified basal medium of Murashige and Skoog (1962) supplemented with either 2 mg/l^-1 2,4-D+15% (v/v) CM or 4 mg/l^-1 2,4-D+2 mg/l^-1 NAA+2 mg/l^-1 KN+1 g/l^-1 YE. Shoot primordia developed after 2–3 subcultures in that medium. Increased growth of shoot primordia was obtained in media containing less auxins and vitamins. Rooted bulbous plantlets obtained were maintained in MS medium with 0.5% sucrose.Adventitious shoots were induced from adaxial epidermal cells of outer scales of regenerated bulbs used as secondary expiants in presence of 1 mg/l^-1 of 2,4-D with slightly higher concentration of the three vitamins of MS medium. From each scale leaf, approximately 400 bulblets were produced in 18 weeks in liquid culture. 90% of the plants transferred to potted soil have survived.     

Gynogenic plant regeneration from unpollinated flower explants of Eragrostis tef (Zuccagni) Trotter

Tef [Eragrostis tef (Zucc.) Trotter] is the most important cereal in Ethiopia. In its wild relative E. mexicana, regeneration of six green plants resulted from culture of 121 non-pollinated immature pistils. In the allotetraploid crop species tef, however, only callus and root formation was obtained by this method. By contrast, immature spikelets and panicle segments of E. tef proved amenable to gynogenic plant regeneration. Upon step-wise optimization of the protocol, efficient plant formation was achieved in all three cultivars tested. In cv. DZ-01-196, culture of 1305 immature spikelets resulted in formation of 159 green plants. Flow cytometric analysis revealed (di)haploid, triploid, tetraploid and octoploid regenerants, from which the vast majority was tetraploid. Tef-breeding programs will likely benefit substantially from efficient generation of true-breeding plants.     

In vitro plantlet regeneration from seedling nodal explants of Acacia catechu

Multiple shoots were initiated after 20 days in stem nodes excised from in vitro grown seedlings of Acacia catechu, on Murashige and Skoog's medium adjuvanted with 1 to 100 microM of N6-benzyladenine (BA). Explants were subcultured on the same medium augmented with 1.5 g l(-1) of polyvinylpyrrolidone (PVP) after 30 days. In the second subculture, after 30 days, the explants were transferred to a medium lacking PVP, but containing 10 microM of BA, where nine or ten shoots differentiated per explant within next 30 days. If individual shoots along with some callus were subcultured on BA (10 microM), nearly 15 shoots per explant regenerated in 90 days. Thus, the average number of shoots obtained from each node was 142 after 180 days. Since a seedling develops four nodes after 20 days, theoretically an average of 568 shoots can be obtained from a single seed. If shoots were individually subcultured on 1/2-strength MS medium with 14.7 microM of indole-3-butyric acid (IBA), roots developed in 20 days. Addition of 40 mg l(-1) of glutamic acid to the rooting medium prevented leaf senescence. These plantlets thrived well in garden soil, sand and silica (1:1:1).     

High efficiency plant regeneration from petiole explants of Carica papaya L. through organogenesis

Callus cultures were obrained from petiole explants of Carica papaya on MS medium containing 0.5–10.5 μM α-naphthaleneacetic acid (NAA) in combination with 0.5–5 μM benzyladenine (BA). Hard-green calli were transferred to MS medium containing 100 mgl⁻¹ casein hydrolysate (CH) with specific BA-NAA formulation, where they developed adventitious buds within 2 weeks of culture. Maximum number of adventitious buds were obtained in 2 μM BA and 0.1 μM NAA. Shoot regeneration occurred from these adventitious buds by the end of the 4th week. Regenerated shoots were elongated in hormone-free medium and rooted in half-strength MS fortified with 3 UM NAA and 0.5 μM gibberellic acid (GA₃). The regenerants were transferred to soil after acclimatization.     

Effect of plant growth regulators on indirect shoot organogenesis of Ficus religiosa through seedling derived petiole segments

Ficus religiosa is known as a long-lived multipurpose forest tree. The tree plays an important role for religious, medicinal, and ornamental purposes. However, the propagation rate of Ficus religiosa is low in natural habitat so the plant tissue culture techniques are an applicable method for multiplication of this valuable medicinal plants. Thus, the aim of this study is to understand the effect of different auxin/cytokinin ratios on indirect shoot organogenesis of this plant. According to our results, the maximum callus induction frequency (100%) was obtained on Murashige and Skoog (MS) medium supplemented with 0.5?mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) plus 0.05?mg/l 6-benzylaminopurine (BAP) from petiole segments. For shoot induction purpose, the yellow-brownish, friable, organogenic calli were inoculated on shoot induction medium. On MS medium supplemented with 1.5?mg/l BAP and 0.15?mg/l Indole-3-butyric acid (IBA), 96.66% of the petiole-derived calli responded with an average number of 3.56 shoots per culture. The highest root formation frequency (96.66%), root number (5.5), and root length (4.83?cm) were achieved on MS medium containing 2.0?mg/l IBA plus 0.1?mg/l Naphthaleneacetic acid (NAA). The rooted shoots were successfully transferred to field condition and the substrate with the mixture of cocopeat and perlite (1:1) had the highest survival rate (96.66%). This is the first report of an effective in vitro organogenesis protocol for F. religiosa by indirect shoot organogenesis through axenic seedling derived petiole explants, which can be efficiently employed for conservation of this important medicinal plant species as well as the utilization of active biomolecules.     

Apical callus formation and plant regeneration controlled by plant growth regulators on axenic culture of the red alga Gracilariopsis tenuifrons (Gracilariales, Rhodophyta)

Axenic cultures of Gracilariopsis tenuifrons (Bird et Oliveira) Fredericq et Hommersand (Gracilariales, Rhodophyta) were established in ASP12‐NTA solid medium (0.4% agar and 1.0% sucrose) supplemented with plant growth regulators to evaluate the effects on apical callus formation and plant regeneration. Indole‐3‐acetic acid (IAA), 2,4‐dichlorophenoxyacetic acid (2,4‐D) and 6‐benzylaminopurine (BA) were added individually or in combinations (IAA : BA) over a range of concentrations from 0.5 to 5 mg L^?1. Growth of apical and intercalary segments was stimulated by high concentrations of 2,4‐D (5 mg L^?1) and a high IAA to BA ratio (IAA : BA = 5:1 mg L^?1) respectively. Apical calluses were originated from divisions of apical and cortical cells located at apical regions of thallus segments and lateral branches. Low concentration of IAA (0.5 mg L^?1) or a high IAA to BA ratio (IAA : BA = 5:1 mg L^?1) were the optimal treatments for inducing apical callus formation in apical segments, while high concentration of IAA (5 mg L^?1) stimulated the highest callus induction rate in intercalary segments. Conversely, equal parts IAA and BA (IAA : BA = 1:1 mg L^?1) and low concentration of 2,4‐D (0.5 mg L^?1) stimulated growth of apical calluses from apical and intercalary segments, respectively. Two processes of regeneration were observed: direct regeneration (upright axis originated from cells of proximal region of intercalary segments) and indirect regeneration (adventitious plantlet originated from cells of apical calluses). Direct regeneration was promoted significantly by treatment with a low IAA to BA ratio (IAA : BA= 1:5 mg L^?1), and treatments with IAA (0.5 mgL^?1) or 2,4‐D (0.5 or 5 mg L^?1) significantly stimulated the elongation of upright axis. Plant growth regulators are essential to inducing indirect regeneration, and a high concentration of IAA (5 mg L^?1) and BA (5 mg L^?1) were the optimal treatments for inducing the regeneration of plantlets from apical calluses in apical and intercalary segments, respectively. Regenerating plantlets grew into plants morphologically similar to those formed from germinating spores, and became fertile after 6 weeks. The results suggest that auxins and cytokinins are involved in developmental regulatory processes in G. tenuifrons. The regeneration process from calluses in species of Gracilariales was observed for the first time in the present study. The culture system described for G. tenuifrons could be useful for micropropagation and for biotechnological applications in agarophytic algae.