Presence Of 6-benzylaminopurine Research Articles

Specific features of <i>in vitro</i> micropropagation of various representatives of the genus <i>Rhododendron</i>

One of the best plants for landscaping urban areas are representatives of the genus Rhododendron L. However, there is the problem of obtaining planting material because traditional propagation methods are inefficient or take a lot of time and resources. In vitro micropropagation can be a solution to this problem. The research objects were Rhododendron Helsinky University, Rhododendron hybrid Marianna v. Weizsacker, and Rhododendron caucasicum var. Rosea. Three variants of Woody Plant Medium (WPM) were used for the experiment: with the addition of benzylaminopurine/indolylbutyric acid or naphthylacetic acid/kinetin in the proportion of mg/l, and a control variant without hormones. The analysis of the effect of different concentrations of phytohormones has demonstrated that for different representatives there are specific combinations of growth regulators. For Rh. caucasicum var. Rosea the hormone-free medium was the most suitable for plant growth as well as the appearance of shoots of order II. For Rh. hybrid Marianna v. Weizsacker, the optimal medium for plant growth was also a hormone-free medium, but the addition of naphthylacetic acid / kinetin enhanced the tillering. The presence of benzylaminopurine / indolylbutyric in the medium had a negative effect on the species and the hybrid. The use of these hormones for Rh. Helsinky University resulted in stimulation of growth and tillering of the plant, while naphthylacetic acid / kinetin hormones caused a depressing effect.

Indirect Somatic Embryogenesis: An Efficient and Genetically Reliable Clonal Propagation System for Ananas comosus L. Merr. Hybrid “MD2”

The objective of this study was to establish an efficient—direct or indirect—regeneration system for pineapple (Ananas comosus L.) plants, with a high rate of multiplication and that would preserve the genetic identity of the donor genotype (Hybrid ‘MD2’) in the regenerated plants. Ten treatments, with different concentrations of 2,4-Dichlorophenoxyacetic (2,4-D) and Picloram (P), in the absence or presence of 6-Benzylaminopurine (BAP), were used for in vitro morphogenesis induction, as well as histological and molecular techniques, in order to characterize the morphogenic responses induced. Significant differences between treatments tested, to induce callus and buds, were assessed by the Kruskal Wallis method and the Mann–Whitney U-tests. Different pineapple regeneration routes were identified, showing the high regeneration potential of this species. The medium containing 2 mg L−1 2,4-D and 2 mg L−1 BAP, where indirect somatic embryogenesis occurred, was selected as the most efficient treatment, with an average of 120 somatic embryos per explant, differing significantly from the rest of the treatments. It was also demonstrated that the pineapple plants regenerated in vitro preserved the genetic identity of the donor genotype, which represents a high degree of confidence for the application of indirect somatic embryogenesis for A. comusus clonal propagation.

Biosynthesis of anti-leishmanial natural products in callus cultures of Artemisia scoparia

Background: Clinically, available synthetic chemotherapeutics in the treatment for leishmaniasis are associated with serious complications, such as toxicity and emergence of resistance. Natural products from plants can provide better remedies against the Leishmania parasite and can possibly minimize the associated side effects. Methods and materials: In this study, various extracts of the callus cultures of Artimisia scoparia established in response to different plant growth regulators (PGRs) were evaluated for their anti-leishmanial effects against Leishmania tropica promastigotes, followed by an investigation of the possible mechanism of action through reactive apoptosis assay using fluorescent microscopy. Results: Amongst the different callus extracts, higher anti-leishmanial activity (IC50: 19.13 mg/mL) was observed in the callus raised in-vitro in the presence of 6-Benzylaminopurine (BA) plus 2,4-Dichlorophenoxyacetic Acid (2,4-D) at the concentration of 1.5 mg/L, each. Further, the results of apoptosis assay showed a large number of early-stage apoptotic (EA) and late-stage apoptotic (LA) cells in the Leishmania under the effect of callus extract grown in-vitro at BA plus 2,4-D. For the determination of the potent natural products in the callus extracts responsible for the anti-leishmanial activity, extracts were subjected to Gas chromatography-mass spectrometry (GC-MS) for the metabolite analysis. Nonetheless, higher levels of the metabolites, such as nerolidol (22%), pelletierine (18%), aspidin (15%) and ascaridole (11%) were detected in the callus grown in vitro at BA plus 2,4-D (1.5 mg/L, each). Conclusion: This protocol determines a novel method of production of anti-leishmanial natural products through callus cultures of A. scoparia, a medicinal plant.

Biosynthesis of anti-leishmanial natural products in callus cultures of Artemisia scoparia

Clinically, available synthetic chemotherapeutics in the treatment for leishmaniasis are associated with serious complications, such as toxicity and emergence of resistance. Natural products from plants can provide better remedies against the Leishmania parasite and can possibly minimize the associated side effects. In this study, various extracts of the callus cultures of Artimisia scoparia established in response to different plant growth regulators (PGRs) were evaluated for their anti-leishmanial effects against Leishmania tropica promastigotes, followed by an investigation of the possible mechanism of action through reactive apoptosis assay using fluorescent microscopy. Amongst the different callus extracts, higher anti-leishmanial activity (IC50:19.13 µg/mL) was observed in the callus raised in-vitro in the presence of 6-Benzylaminopurine (BA) plus 2,4-Dichlorophenoxyacetic Acid (2,4-D) at the concentration of 1.5 mg/L, each. Further, the results of apoptosis assay showed a large number of early-stage apoptotic (EA) and late-stage apoptotic (LA) cells in the Leishmania under the effect of callus extract grown in-vitro at BA plus 2,4-D. For the determination of the potent natural products in the callus extracts responsible for the anti-leishmanial activity, extracts were subjected to Gas chromatography-mass spectrometry (GC-MS) for the metabolite analysis. Nonetheless, higher levels of the metabolites, such as nerolidol (22%), pelletierine (18%), aspidin (15%) and ascaridole (11%) were detected in the callus grown in vitro at BA plus 2,4-D (1.5 mg/L, each). This protocol determines a novel method of production of anti-leishmanial natural products through callus cultures of A. scoparia, a medicinal plant.

Indoleamines and phenylpropanoids modify development in the bryophyte Plagiomnium cuspidatum (Hedw.) T.J. Kop

An efficient method for in vitro propagation of the bryophyte moss Plagiomnium cuspidatum (Hedw.) T.J. Kop is presented. Protocol optimization investigated media salt strength (quarter, third, or half-strength Murashige and Skoog; MS), sugar concentration (1 to 3% (w/v) sucrose), growth regulator content (presence of benzylaminopurine (BA) at 0–5 μM and napthaleneacetic acid (NAA) at 0–5 μM), and the addition of the phenylpropanoid biosynthesis inhibitor 2-aminoindan-2-phosphonic acid (AIP). Optimal media composition was determined to be half-strength MS with 2% (w/v) sucrose and 0.1 μM NAA. This method was then utilized to examine the effects of modified phenylpropanoid metabolism via application of AIP, an inhibitor of the first dedicated enzyme in phenylpropanoid biosynthesis, phenylalanine ammonia lyase (PAL). Treatment with AIP greatly reduced tissue browning and initiation of branching in P. cuspidatum and resulted in prolific production of straw to pale green-colored rhizoids. Treatment of plants with AIP in combination with the biosynthetic product of PAL, trans-cinnamic acid, was not able to fully recover the branching or browning phenotype, but several other phenolics, including p-coumaric acid and kaempferol, produced farther downstream in the biosynthetic pathway, were capable of partial recovery of the phenotype. Additionally, treatment with two indoleamines, melatonin and its biosynthetic precursor N-acetylserotonin, was also capable of partial recovery of the phenotype, showing greatly increased branching and increased browning of rhizoids. These results suggest that cross talk between phenylpropanoid and indoleamine metabolism is involved in bryophyte growth and development, beyond their traditional roles, leading to modified developmental outcomes.

UV-C Stresinin 6-Benzilaminopürin Varlığında Çoğaltılmış Amsonia orientalis’in In vitro Sürgün Gelişimi ve Antioksidan Enzimleri Üzerine Etkileri

Bu calismada abiyotik faktorlerden biri olan UV-C stresinin 6-benzilaminopurin varliginda cogaltilmis Amsonia orientalis’in in vitro surgunleri uzerindeki etkileri, surgunlerin morfometrik ozellikleri, H2O2 (hidrojen peroksit) ve MDA (malondialdehit) seviyeleri ile antioksidan enzimleri analiz edilerek irdelenmistir. Test edilen 3,47 (15 dk), 6,94 (30 dk) ve 13,87 (60 dk) kJ m-2’lik UV-C radyasyon dozlarinin hepsi surgun uzunlugu ile surgun sayisinda kontrole (UV-C uygulanmamis bitki) oranla dususe sebep olmustur. Radyasyon kaynakli olusan bu stres 6-benzilaminopurin varliginda surgun gelisimini yavaslatsa da tamamen durdurmamistir. UV-C radyasyon uygulamalari antioksidan enzimler uzerine farkli etkiler gostermistir. SOD (superoksit dismutaz) aktivitesi artan radyasyon dozuna bagli olarak kontrole gore onemli olcude azalirken, POD (peroksidaz) aktivitesinde artis gozlenmistir. CAT (katalaz) aktivitesinin ise radyasyondan onemli olcude etkilenmedigi gorulmustur. Artan UV-C dozlarinda H2O2 ve MDA seviyelerinin benzer olarak arttigi bulunmustur. Elektroforetik analizler sonucunda SOD enzimi icin uc farkli izozim (Cu/Zn-SOD, Fe-SOD ve Mn-SOD) tespit edilmis olup, artan UV-C stresinin bitkinin Cu/Zn-SOD aktivitesini tamamen inhibe ettigi gozlenmistir. Sonuclar bitkinin UV stresine karsi gosterdigi savunma mekanizmasinda peroksidazlarin incelenen diger antioksidan enzimlere gore oncelikli rol aldigina isaret etmektedir.

Imposed starvation for in vitro maintenance and high regeneration of elite key lime (Citrus aurantifolia)

ABSTRACTPreservation of elite key lime (Citrus aurantifolia) genotypes using efficient micropropagation and regeneration protocols is very important for new breeding programmes. The objective of the present study was to develop an efficient protocol for micropropagation and maintenance of elite genotypes of key lime based on slow growth and starvation strategy. To do this, regenerated shoots were starved, and the growth was compromised via avoiding subcultures for 4, 8, 12 and 24 months. The numbers of responsive explants and adventitious shoots per each explant were evaluated in the presence of benzylaminopurine (BAP). The results showed that the 12- and 24-month starved explants treated with 1 mg/l BAP were able to produce four times more shoots than the control. In addition, the influences of horizontal or vertical layering explants over medium were evaluated. The results indicated that all horizontally layered explants significantly produced 25% adventitious shoots more than vertical explants, most likely owing to more contact with the surface medium. Finally, it could be concluded that application of slow growth, starvation strategy and also horizontally layering of the explants could efficiently enhance micropropagation and maintenance of threatened elite key lime genotypes with a low cost.

An Evaluation of Banana Macropropagation Techniques for Producing Pig Fodder in Northern Thailand

Smallholder farmers raising pigs in northern Thailand rely heavily on banana stalks as a fermented feed source, but struggle to reproduce banana plants fast enough to keep up with consumption. This study evaluated a variety of techniques for rapidly multiplying banana plants, using techniques appropriate and affordable to smallholder farmers in order to help meet this demand. Propagation techniques of Musa (ABB) cv. ‘Kluai Nam Wa’ were conducted in greenhouse and field experiments in both lowland and upland areas of Chiang Mai Province, Thailand. In greenhouse experiments, six treatments were conducted during the dry and rainy seasons, while five different treatments were compared in the field. Treatments used various methods of mechanical injury or application of benzyl aminopurine (BA) to induce plantlet differentiation. Number of plantlets to emerge, days to emergence, and circumference of plantlets were observed over a 90-day period. Results indicate that time of year plays an important role in the macropropagation of bananas, as significantly higher numbers of plantlets emerged during the rainy season. Plantlets emerged in 65 days, on average, during the dry season, but took only 54 days during the rainy season. During the rainy season, the presence of BA produced more plantlets than the other treatments, but during the dry season, there were no differences among treatments. Overall, the number of plantlets produced in all treatments evaluated was very low; however we believe this research is an important contribution to the literature and acknowledge that there exists significant opportunity to capitalize on the low-cost appropriate technology benefits that macropropagation of bananas can deliver to smallholder farmers.

Large-scale Micropropagation of Aloe vera

A protocol for large-scale Aloe vera (L.) Burm. f. production was established using micropropagation of apical buds. The effects of two chlorine-based disinfectants were evaluated on the survival of the explants in different treatments in a semisolidified Murashige and Skoog (MS) medium in the presence of 6-benzylaminopurine (6-BAP; 2 mg·L−1). During 120 days, 136 green apical shoots bearing axillary buds were multiplied four times at 30-day intervals in the same MS medium, reinoculating seven to nine explants per flask each time. The elongation and rooting processes were carried out in the same MS medium without 6-BAP. A total of 40,495 Aloe vera microplants were obtained, a yield of 300 microplants per apical bud at a rate of 1:5.3 in every multiplication period of 30 days. From that total, 38,480 Aloe vera microplants were successfully acclimatized transferring to 36- and 64-cell polyethylene trays containing proper substrate in two different ex vitro greenhouse conditions. After a 3-month period, fresh and dry matter weights of the Aloe vera plants were determined. All the data from each experimental phase were statistically analyzed. The use of 64-cell (40 cm3/cell) trays represented an economy of 47.37% in greenhouse space and 50% in the amount of substrate per Aloe vera plant.

Secoiridoid content of Blackstonia perfoliata in vivo and in vitro

This study reports the analysis of secondary metabolites of gentiopicrin, swertiamarin, and sweroside in shoot and root cultures of yellow wort (Blackstonia perfoliata), which were initiated from seeds, grown on Murashige and Skoog (MS) medium. Shoot cultures of B. perfoliata were inoculated with suspension of Agrobacterium rhizogenes strain A4M70GUS and hairy roots appeared at the infected sites after 3 wk of inoculation. Tips of adventitious roots of B. perfoliata were grown on hormone-free MS medium and three clones of the transformed roots regenerated shoots spontaneously. Gentiopicrin, swertiamarin, and sweroside were detected in both roots and shoots of B. perfoliata in vitro and in vivo, but gentiopicrin was found to be the major compound. The concentration of growth regulator in the medium affected the production of secoiridoids in B. perfoliata in vitro, where the level of gentiopicrin was higher in plants grown in the presence of indole-3-butyric acid, but the presence of 6-benzylaminopurine was inhibitory to secoiridoid production.

In vitro culture of Pandanus amaryllifolius and enhancement of 2‐acetyl‐1‐pyrroline, the major flavouring compound of aromatic rice, by precursor feeding of L‐proline

AbstractShoots, plantlets and semi‐differentiated callus (SDC) cultures of Pandanus amaryllifolius capable of producing high levels of basmati rice flavour were established in vitro using Murashige and Skoog nutrient medium. A total of 10% of the initial explants responded to produce shoot cultures in the presence of benzylamino purine (BAP) (0.5 mg L−1) and glutamine (100 mg L−1). Leaf explants and basal portions of shoots produced SDC whereas elongated in vitro shoots could be continuously multiplied, using BAP (1.5 mg L−1) and kinetin (Kn) (1.0 mg L−1), and rooted in half‐strength medium for ex vitro cultivation leading to a process of micropropagation. Steam‐distillation extraction (SDE) followed by gas chromatography‐mass spectrometry (GC‐MS) analysis of various cultured organs and spent liquid medium used for SDC revealed the presence of 2‐acetyl‐1‐pyrroline (2‐AP) to various extents. This 2‐AP compound has been identified as the major flavouring compound of scented basmati and other scented rice varieties. 2‐AP was found to be highest, on a fresh weight basis, in SDC (19.7 mg kg−1) on the 40th day, whereas in vitro roots, shoots and field leaves (of one‐year‐old plant) had lower levels of 15, 6.8 and 14 mg kg−1, respectively. Further enhancement of 2‐AP in SDC using precursor was possible by feeding into medium 1 mmol L−1 of L‐proline where a highest level of 21.67 ppm of 2‐AP accumulated on the seventh day whereas a higher level of 2 mmol L−1 of L‐proline suppressed 2‐AP levels. The present report is the first on the tissue culture studies of P. amaryllifolius where continuous production of plantlets as well as synthesis of high levels of 2‐AP has been documented. Copyright © 2005 Society of Chemical Industry

In vitro Regeneration and Transformation of Blackstonia perfoliata

In vitro root culture of yellow wort (Blackstonia perfoliata (L.) Huds.) was initiated on Murashige and Skoog (MS) medium. In the presence of benzylaminopurine (BAP) numerous adventitious buds formed, which developed into shoots. Presence of indole-3-butyric acid (IBA) in media significantly decreased number of buds, but increased development of lateral roots. On hormone-free medium shoots successfully rooted and developed flowers and viable seeds that formed another generation. Shoot cultures of B. perfoliata inoculated with suspension of Agrobacterium rhizogenes strain A4M70GUS developed hairy roots at 3 weeks and they were cultured on hormone-free MS medium. Spontaneous shoot regeneration occurred in 3 clones.

Relationship between Salinity-Induced Damages and Aging in Rice Leaf Tissues

Segments of rice leaves at different nodal positions were incubated in NaCl solutions for various periods, and the chlorophyll content, Na content, CI content, Na/K ratio and the ultrastructure of excised leaf tissues were examined. The chlorophyll content of the leaf tissues decreased with increasing NaCl concentration and incubation period. Na and CI contents of the leaf tissues also increased with increasing NaCl concentration, but the decrease in chlorophyll content by salt stress was greater in old than in young tissues even when both tissues contained comparable amounts of Na and CI. The presence of benzylaminopurine (BAP) alleviated the salt stress-induced decrease in chlorophyll content, but did not significantly affect the element contents. Ultrastructural damages were apparent in the chloroplasts of the leaf tissues subjected to salt stress. In 0.1% NaCl-treated old leaf tissues, the thylakoids were swollen, the envelope was partly destroyed causing leakage of the chloroplast contents. However, these damages were alleviated by the addition of BAP to the NaCl solution. In young leaf tissues, the thylakoids were swollen by incubation in 1.0% NaCl solution, but no structural distortion was observed in a 0.1% NaCl solution even without BAP added. The present study suggests that the leaf tissues were damaged by an increasing salt content and became more sensitive to salt stress with advancing leaf age. BAP seemed to alleviate the damages by salt stress through retardation of leaf aging.

Restorative Morphogenesis in vitro in the Annual Medic in the Presence of Benzylaminopurine

Four annual medic species (Medicago orbicularis (L.) All., M. rigidula (L.) Desr., M. scutellata (L.) Miller, and M. rugosa Desr.) were used as model objects for studying the spectrum of morphogenetic reactions in vitro. The seeds were incubated on nutrient media with benzylaminopurine at different concentrations until germination and, thereafter, the explants of seedlings were cultivated in order to obtain primary calluses and morphogenetic structures. Normal and abnormal (with reduced root and/or apex) seedlings were cultivated in the presence of benzylaminopurine. Further cultivation of explants from the seedlings of both types showed a considerable intra- and interspecific polymorphism by the capacity for callusogenesis, frequency of primary restorative reactions, and pattern of microreproduction in vitro. In the control (the seeds were incubated on a hormone-free medium), no cases of microreproduction by way of organogenesis or somatic embryogenesis were observed. In all experimental variants, the restorative reactions preceded microreproduction in vitro.

In vitro propagation of Leptospermum hybrids

Tissue culture protocols were developed for superior ornamental Leptospermum hybrids. Benzylaminopurine (BAP) was the most effective cytokinin for the proliferation of shoots. On modified Murashige & Skoog (MS) media with low BAP concentrations (0.05–0.1 mg litre−1), an average of up to nine shoots developed from single nodes, although there were significant differences between the clones. Mean shoot length was relatively short for all clones growing in the presence of BAP. Adding gibberellic acid (GA3) to the proliferation medium increased shoot length only at the highest rate assessed (10 mg litre−1), but this resulted in shoots that were spindly and chlorotic and reduced in number. A reduction in the culture room light level slightly reduced shoot length. Shoots developed roots, either on a growth regulator‐free medium, or on a medium with low concentrations of indole‐acetic acid (IAA) or indolebutyric acid (IBA). Rooted plantlets were successfully transferred to the greenhouse environment.

Tapioca-a new and cheaper alternative to agar for direct in vitro shoot regeneration and microtuber production from nodal cultures of potato.

The efficiency of in vitro shoot regeneration and microtuber production of potato (Solanum tuberosum L.) from nodal explants was studied using agar and other new and cheaper gelling agents- tapioca and sago in Murashige & Skoog (MS) salt medium. For shoot regeneration, agar was maintained at 8 mg l -1, tapioca and sago were varied between 9-18 and 10-14 % (w/v), respectively supplemented with 3% sucrose, 0.03 mg l-1 NAA, 0.25 mg l-1 GA3 and 2.5 mg l-1 Ca-panthothnate. The microtuberisation study was done using agar, tapioca and sago at 0.8, 14 and 10% concentration (w/v), respectively, in the presence of benzylaminopurine and paclobutrazol. Type of gelling agent significantly affected in vitro plant regeneration. After 32 days, shoot height and number of nodes respectively were 8.86 cm and 10.5 in agar, and 8.9 -12.1 in 11-15% tapioca, respectively. Tapioca at 14-15% stimulated growth giving significantly (P

Attempt at regeneration of Pharbitis nil from fragments of vegetative organs

The object of study was the regeneration of Pharbitis nil by direct and indirect organogenesis. From fragments of roots, cotyledons, hypocotyls and epicotyls on Murashige and Skoog nutrient solution (MS) supplemented with naphtalenacetic acid (NAA) or indolylacetic acid (IAA; both at 0.1 mg·dm−3 concentration) in the presence of benzylaminopurine (BAP), zeatin or kinetin (all at 5 mg·dm−3 concentration) only root organogenesis was obtained. Likewise, when using the two-step method (2 or 5 days exposure to NAA or IAA at 2 mg·dm−3 concentration followed by exposure to BAP or zeatin at 1 or 2 mg·dm−3 concentration) root organogenesis was observed in all types of explants. Moreover, shoot buds were formed on fragments of epicotyl exposed vertically in relation to the medium. However, attempts at regenerating complete plants from them failed, so did the regeneration of P. nil from callus. The roots were formed in callus cultures only.

Rapid multiplication of Phyllanthus fraternus: a plant with anti-hepatitis viral activity

Aqueous extracts of Phyllanthus fraternus (used in Ayurveda and Siddha systems of medicine for the treatment of jaundice) possesses anti-hepatitis virus DNA polymerase activity. As the availability of P. fraternus is restricted by geographical distribution and seasonal growth, the present investigations were aimed at standardizing procedures for micropropagation of P. fraternus using shoot tip and nodal cultures to make it available all-year-round. Shoot tip and nodal explants were cultured on B 5 medium supplemented with 0, 10 −7, 10 −6 and 10 −5 M benzylaminopurine (BAP) or kinetin (KN). The presence of BAP in the medium promoted multiple shoot formation from both types of explants. Of the two explants, shoot tips were found to be more amenable for shoot multiplication. About 14–16 shoots were obtained from a single shoot tip in approximately 28 days on medium with 10 −5 M BAP. Whenever required the individual shoots could be rooted on half-strength B 5 medium supplemented with 10 −6 M IBA. Thus the media standardised for multiple shoot formation/axillary bud sprouting through shoot tip cultures can be exploited for multiplication of desirable genotypes or potential somaclonal variants (such as those with high levels of active principle or with higher therapeutical value) at commercial level. Moreover, these can be directly used for drug production without any chemical pretreatments.

Canavanine metabolism in tissue cultures of Canavalia lineata

The greening of callus was achieved by modulating the medium's growth regulator concentrations under continuous light. Canavalia lineata (L.) DC. calluses formed chlorophyll when they were exposed to continuous light in the presence of benzylaminopurine and indole-3-acetic acid. Canavanine and canaline were detected in the green callus. But only canaline was detected in the white callus grown in the dark. Feedings of canaline to suspension cultures showed that the green suspended cells were capable of de novo biosynthesis of canavanine, but the white suspended cells were not. Exogeneously supplied canavanine was used to produce canaline and homoserine by the white suspended cells. Arginase activity was induced by the addition of arginine or canavanine to the medium, and canaline reductase activity was induced by the addition of canaline but not with ornithine in the white suspended cells.

Plant regeneration by organogenesis in Vitis rootstock species

A procedure for the regeneration of Vitis rootstocks plantlets by organogenesis from foliar tissues is described. Leaves from mature plants grown in growth chambers or from plantlets grown in tubes were wounded with a scalpel and cultured on a modified Murashige and Skoog liquid medium containing different concentrations of benzyl-aminopurine. The presence of benzyl-aminopurine is required for shoot formation. The age of the source explant, the composition of the culture medium and the culture temperature are important parameters of the regeneration process.