Addition Of Growth Regulators Research Articles

IN VITRO MORPHOGENESIS OF GYPSOPHILA: RESPONSE TO EXOGENOUS PHYTOHORMONES

IN VITRO MORPHOGENESIS OF GYPSOPHILA: RESPONSE TO EXOGENOUS PHYTOHORMONES

Micropropagation of Bulbophyllum Orchids

Abstract— Bulbophyllum (Orchidaceae) is recognized as the largest genus of orchids in the world. Bulbophyllum is a popular orchid by its unique flowers and its benefits. Although Bulbophyllum is a large genus, research on in vitro propagation of orchids from this genus is still limited. This article aims to examine the micropropagation of several Bulbophyllum species to determine the affecting factor for the propagation of Bulbophyllum orchids. A systematic Literature Review (SLR) uses in this paper. The micropropagation of Bulbophyllum have studied in B.auricomum, B. fascinator, B. macrantum, B. phalaenopsis, B. lasianthum B. odoratissimum B. niphondii, B. dhaninivatii, B. lilacinum, B. echinolabium, B. putidum and B. plumatum. The use of appropriate media can increase the growth of orchids. The addition of growth regulators and/or organic compounds to a suitable basal medium can be a way to find a suitable medium for orchid growth. Acclimatization should be conducted on plantlets with healthy roots. The appropriate acclimatization media can increase the survival rate of orchid seedlings.

Effect of Abiotic and Biotic Elicitors on Callus and Suspension from Piper Betle L. Var. Nigra

Piper betle L. var. Nigra (black betel) contains secondary metabolites and has biological activity as antibacterial, antifungal, antioxidant, etc. To increase the production of secondary metabolites, an alternative method is needed, namely cell suspension culture. This study aims to determine the effect of abiotic and biotic elicitor on callus biomass produced from cell suspension culture. Leaf explants were grown on Murasige and Skoog (MS) medium with the addition of growth regulator 2.4-D 0.5 mg/L and BAP 2.0 mg/L with abiotic elicitors CuSO4, ZnSO4, HgCl2 and CoCl2 with a concentration of 0.5; 1.0 and 2.5 mg/L. The biotic elicitor used was Aspergillus niger with a concentration of 0.025%; 0.050% and 0.1%. The cultures were incubated for 8 weeks. 0.5 g callus was subcultured on 25 mL cell suspension medium. The suspension culture was shaken at 110 rpm. In this suspension culture, the callus was incubated for 3 weeks. After 3 weeks of age callus on suspension medium was harvested and weighed the fresh and dry weight. The results showed that the highest average dry weight was found in the treatment with abiotic elicitor CuSO4 0.5 mg/L at 0.088 g.

Micropropagation of bog blueberry (Vaccinium uliginosum L.) distributed in Bulgaria

The aim of the present study is to develop a system for micropropagation of bog blueberries. They are distributed from 1700 to 2500 m above sea level in the mountains Rila, Pirin, Western and Central Balkan Mountain, Vitosha, Osogovo, Western Sredna Gora and Western and Middle Rhodopes. An approach for the statistical optimization of micropropagation media under laboratory conditions has been applied for the first time. Sterile explants were introduced in vitro on WPM, enriched with 3 mg.l-1 2-iP and pH 4.2. To optimize proliferation, nine variants of WPM were tested with the addition of growth regulators (zeatin and 2-iP) in varying concentrations (1.5 - 3.5 mg.l-1), organic additives (casein hydrolisate and myo-inositol and pH values (4.3 - 4.8). The best result regarding the reproduction coefficient was found on M1 nutrient medium (3.77 pcs/explant), enriched with 3.5 mg.l-1 zeatin and 2-iP, 250 mg.l-1 casein hydrolisate, pH 4.8. The largest average length of shoots was reported on the modified medium M9 with 2.63 cm. The resulting micro shoots were rooted on WPM nutrient medium, halved macrosol content and different levels of IAA (0.5 - 1 mg.l-1), myo-inositol and pH. The highest percentage of rooting was found on R8 nutrient medium with 56.7%.

Ratio of Yeast Extract Administration on Induction in vitro planlet black orchid (Coelogyne pandurata lindl) From Kalimantan

Black Orchid (Coelogyne pandurata) is a flora from Kalimantan that has uniqueness and beauty on the tongue of its flowers. Plants multiply naturally, non-natural propagation has many obstacles, difficult crossing. Plant breeding efforts are required. Tissue culture techniques are expected to be carried out, several endogenous, exogenous factors and the addition of growth regulators (ZPT) are expected to be research efforts for the propagation of this orchid. Efforts to add yeast extract are expected to be a source of growth regulators, because yeast is rich in proteins, free amino acids, peptides, nucleotides, and complex vitamins. This study used the addition of Yeast Extract to the Induction of in Vitro Planlet Black Orchid ( Coelogyne pandurata Lindl) 0.5 g / l, 1 g / l and 1.5 g / l. test analysis using SPSS with Kruskal-Wallis nonparametric test analysis, the results showed that the administration of yeast extract had a significant influence on the number of shoots and the number of roots. A concentration of 1 g/l has an influence on the number of shoots (4.33) and the number of leaves (8.00). A concentration of 1.5 g/l of yeast extract affects the number of roots (3.00).

Somatic embryogenesis and plant regeneration from radicles of olive (Olea europaea ‘Chemlal’) zygotic embryos

Olive improvement by biotechnological tools such as genetic transformation requires an efficient in vitro regeneration system. Somatic embryogenesis seems the most suitable process. Our work describes for the first time the regeneration of whole plants in the main olive cultivar in Algeria ‘Chemlal’ via somatic embryogenesis induced from radicles of mature zygotic embryos. The obtained results showed that the establishment of a competent embryogenic culture is highly influenced by the chemical composition of the calli induction and maintenance media as well as addition of growth regulators. More than 10 and 13 % of nodular calli were obtained after callogenesis respectively on MS and OMc solid media containing IBA and zeatin followed by transfer to the same media without zeatin and a reduced concentration of auxin, while embryogenesis rates of 3.3 and 6.7 % were obtained respectively with IAA on MS medium and NAA on both tested media. However, no embryogenesis was observed with 2, 4-D or control which induced less callogenesis. Subsequently, an ECO medium with IBA, zeatin and BA particularly in liquid culture, allows better calli proliferation and embryogenic expression compared to OM and MS media. Finally, matured somatic embryos germinate quickly on a solid OM basal medium and generate normal well-developed plantlets easily acclimatized to natural conditions.

Effect of Media Variation on the Induction and Phytochemical Profile of Callus in Two Varieties of Cat's Whiskers (Orthosiphon aristatus Blume Miq)

The levels of rosmarinic acid and sinensetin in purple and white-purple varieties of Orthosiphon aristatus, cat's whiskers, can be increased using modified in vitro culture. This work focused on callus induction of the purple and white-purple variety of cat's whiskers grown on Gamborg (B5) and CHU (N6) with the addition of growth regulators 2,4-dichlorophenoxyacetis acid. Our observation suggested that the callus could grow within three weeks and produce rosmarinic acid and sinensetin. The level of sinensetin from various extraction methods is relatively low; in contrast, the rosmarinic acid from purple callus was detected at about 5% w/w, while the white-purple variety was around 2% w/w. The results of this study also provided new information on the basic media other than MS that can grow cat's whiskers callus while producing active compounds.

The Influence of Various Growth Regulators on Induction Organogenic Callus from Gajah and Kuning Cassava Genotype (Manihot esculenta Crantz)

Kuning and Gajah genotypes are two collections of cassava in the Biotechnology Research Center for Germplasm, LIPI with the advantages of each genotype are high beta carotene and high production. The multiplication in in vitro culture can be done one of them through organogenesis. The aim of this study was to evaluate the effect of using 2,4-D; NAA and Kinetin are used singly for the formation of organogenesis of cassava in the Kuning Cassava and Gajah genotypes. This research was conducted at the Laboratory of Molecular Genetics and Modification of Plant Biosynthetic Pathways, Biteknologi Research Center, LIPI, Bogor since January - February 2018. The source of explants were young leaves and petiols from cassava plant culture in vitro genotypes of Gajah and Kuning yam which were three months old. in culture. The basic media used as a planting medium were Murashige and Skoog (MS) media with the addition of growth regulators (ZPT) singly, 2,4-D, NAA and Kinetin with two concentrations of ZPT each, 8 and 10 mg L- 1 This research was arranged based on a completely randomized design factorial pattern consisting of 2 factors. All data obtained were analyzed using ANOVA and if there is an influence then proceed with the DMRT test with an error rate of 5% using the SPSS program. The highest number of Kuning genotype cassava organogenic callus that developed into shoots on the medium added by ZPT was 2.4 D and kinetin with the same concentration of 8 mg L-1. Formation of the best organogenic callus in petiol explants in the media with the addition of a single 2,4-D and Kinetin with the same concentration of 8 mg L-1. Keywords: Cassava, growth regulators, organogenic.

Effect of Indole 3-Acetic Acid (IAA) and 6-Benzyl Amino Purine (BAP) on Nannochloropsis sp. culture growth

One of the factors influencing the growth of Nannochloropsis sp. is the composition of culture media. The addition of growth regulators in the form of auxins and cytokines in culture media can increase the growth of microalgae. This study aims to examine the effect of IAA (auxin) and BAP (cytokinin) with various concentrations on biomass, chlorophyll content and carbohydrate content of Nannochloropsis sp. culture. Nannochloropsis sp. culture was treated with IAA and BAP in concentration variations each consisting of 0, 0.1, 1, 10 ppm with 3 replications. Data were analyzed with two way ANOVA at 95% confidence level and Tukey follow-up test. The results showed that the combination treatment of IAA and BAP did not affect the chlorophyll-a content of Nannochloropsis sp. culture, but it affected the biomass with the highest P16 (I10B10) of 3.65 g/L and carbohydrate content with the highest content in P4 (I10B0) of 0.30 mg/L. The highest chlorophyll-a content was found in P15(I1B10) of 5.574 mg/L, increased by 6 % compared to controls. Whereas the lowest chlorophyll-a content was found in P12(I10B1) of 1.563 mg/L, decreased by 70 % compared to the control.

Сравнение процесса дыхания у донорных растений озимой пшеницы сорта Иркутская и каллусов, полученных из данного сорта в культуре изолированных пыльников

Winter wheat is one of the main agricultural crops in the world. The constantly growing needs for food, as well as changing climatic conditions require modern breeding of new, more productive varieties with a high degree of cold, frost, drought and salt resistance,and resistance to pathogens and pests. Experimental haploidy can significantly reduce the time required for obtaining pure lines and new varieties. It is known that the physiological state of donor plants is very important for the effectiveness of androgenesis in vitro. In this work, we studied the features of flowers respiration in donor winter wheat plants used to isolate anthers and calli obtained in this culture. Winter wheat of Irkutskaya variety was used in the work. The plants were grown at the artificial climate station of Siberian Institute of Plant Physiology and Biochemistry SB RAS and in the field conditions of the Zalarinsky agroecological station. The plant material was selected at the booting stage. The initial medium was modified N6 containing sucrose (90 g/L), glycine (2 mg/L), 2,4-D (1 mg/L), and NAA (1 mg/L). The medium for regeneration was 190-2Cu with the addition of growth regulators 2,4-D (0.5 mg/L) and kinetin (0.5 mg/L). The oxygen uptake rate was measured at 26 ° C using an Oxyterm system (Han-satech Instruments, Great Britain). To determine the contribution of the main cytochrome and alternative cyanide-resistant pathways to respiration, an inhibitory assay was used. It was shown that opening of the flag leaf, as well as the procedure of anther isolation, led to an increase in the respiratory activity of winter wheat flowers, mainly due to the activation of the cytochrome pathway. At the same time, long-term low-temperature exposure contributed to the stabilization of respiration at the control level and did not cause changes in the activity of the alternative cyanide-resistant oxidase. The maximum respiratory activity was recorded in rhizogenic calli, while the respiratory activity of gemmorhizogenic calli was similar in respiration rate and contribution of the cytochrome and alternative pathways to the respiration of anther donor flowers.

Plant Growth Regulators in the in Vitro Cultivation of Acmella oleracea (L.)

Acmella oleracea is a tropical plant, typical of the northern region of Brazil. The species belongs to the Asteraceae family and has great therapeutic, pharmacological and industrial potential. A limiting factor for the production of this species on a large scale is the short life cycle. The tissue culture programs use synthetic hormones based on cytokinins, such as kinetin and benzylaminopurine (BAP) and auxins such as naphthalene acetic acid (ANA). The objective of this research was to evaluate the effect of growth regulators on the production of Acmella oleracea "in vitro". The experimental test was carried out with control (C), without the addition of growth regulators and five treatments, composed of: (T1) 0.1; (T2) 0.3; (T3) 0.5 mg L-1 kinetin; (T4) 0.1 mg L-1 of BAP and ANA; (T5) 0.5 mg L-1 of BAP and ANA. The experimental design was a completely randomized block in a factorial arrangement with six treatments, three blocks and twenty-five repetitions per block. The evaluated parameters were: germination, root formation, aerial part length, root length, aerial part fresh mass and root fresh mass, aerial part dry mass and root dry mass. The data obtained were subjected to analysis of variance (p <0.05) and compared using the Tukey test. The results showed that kinetin positively contributed to seed germination and aerial part dry mass development. Treatment 1 had the best results for the parameters root length, shoot length and root dry mass.

Особливості введення Pinus strobus L. у культуру in vitro

Досліджено особливості клонального мікророзмноження рослин P. strobus у культурі in vitro. Наведено результати досліджень з підбору реагентів, їх концентрації та експозиції з метою отримання стерильних експлантів, для подальшого їх культивування. Вважають, що найбільше ураження рослинного матеріалу відбувається в літній період, а найменше – у зимовий. У ролі експлантів використано апекси верхівкових бруньок, що заготовляли у другій половині грудня та насіння поточного року. Для стерилізації відібрано зразки, які почергово занурювали в дезінфікувальні розчини гіпохлориту натрію, перекису водню та етилового спирту з різною експозицією. Встановлено, що для успішного введення P. strobus у культуру in vitro найкраще використовувати 2,5 % гіпохлорит натрію з експозицією 6–10 хвилин. Виконано підбір оптимального середовища для розмноження рослинного матеріалу та підвищення частоти регенерування – частки експлантів, які утворили адвентивні мікропагони та кількості нових мікропагонів на експлант. Бруньки у ролі екслантів не використовували, оскільки після проведення стерилізації їх заражуваність інфекцією була вищою, а відсоток життєздатності нижчий, ніж у насіння. Виявлено, що на середовищі Ллойда-Мак-Коуна та Шенка-Хільдебранта експланти розвивались, але бічні пагони утворювались слабо і майже не росли. На середовищі Гамборга материнські експланти взагалі не розвивались і з часом почали гинути. Найсприятливішим для культивування експлантів виявилося середовище Мурасіге-Скуга, тому подальші дослідження виконували на його основі. Для цього новоутворені експланти, отримані після першого культивування, пересаджували на це середовище, модифіковане додаванням регуляторів росту – індолілоцтової (ІОК) та нафтилоцтової кислот (НОК) з концентрацією 0,5 мг/л та бензиламінопурину (БАП) з концентрацією від 0,5 до 1,5 мг/л. Усього було дев'ять варіантів досліду. Встановлено, що найефективнішим був варіант, у якому в середовище додавали 0,5 мг/л НОК і 1 мг/л БАП.

Bioactive compounds from callus culture of Elaeocarpus grandiflorus

E. grandiflorus has potential as a source of bioactive compounds. This study aims to analyze the content of flavonoid and phenolic bioactive compounds in the callus culture of E. grandiflorus in various concentrations of PGR. Callus culture induction was carried out by maintaining E. grandiflorus leaf stalk explants on solid Murashige & Skoog (MS) with the addition of growth regulators namely 2,4-dichlorophenoxyacetic acid (2,4-D) and picloram in different concentrations. The results of the study showed that callus maintained on MS medium with the addition of 2,4-D and picloram could produce flavonoids and phenolics. Flavonoid and phenolic concentrations produced in each treatment varied. Overall, it can be concluded that the MS medium with additional growth regulators, 2,4-dichlorophenoxyacetic acid (2,4-D) or picloram can be used for bioactive compound production of callus Elaeocarpus grandiflorus.

Production of the secondary metabolite catechin by in vitro cultures of Camellia sinensis L.

Background Catechin is one of the secondary metabolites in Camellia sinensis L. that is alternatively produced through in vitro cultures. The in vitro culture product is possibly improved by optimizing the culture medium with the addition of growth regulators and precursors. The purpose of this study was to confirm the success of the secondary catechin metabolite production through the in vitro culture of C. sinensis L in a relatively short time. Methods The secondary catechin metabolite product is obtained in about 40 days. The study was conducted by (1) leaf cutting for inoculation in Murashige and Skoog media with 1 μg/mL of 2,4-dichlorophenoxyacetic acid growth regulator; (2) the inoculation of callus multiplication on the same medium as a partially modified inoculation media condition with the addition of 1 μg/mL of 6-benzylaminopurine (BAP) and 2 μg/mL of 2,4-dichlorophenoxyacetic acid at concentration; (3) callus multiplication developed on a new medium containing phenylalanine precursors (300 μg/mL); (4) testing growth by harvesting the callus and weighing the wet weight of its biomass and (5) identification of the callus qualitatively and quantitatively by using high-performance liquid chromatography (HPLC). Results The level of secondary catechin metabolite produced was 2.54 μg/mL and 12.13 μg/mL in solid and suspension media, respectively. Conclusions It is concluded that the method is effective and efficient in producing catechin product from C. sinensis L.

Callus Induction of Mangosteen (Garcinia mangostana L.) In Vitro with Addition of Growth Regulators

This research was conducted to determine the best combination of plant growth regulators on callus induction of shoot explants mangosteen (Garcinia mangostana L.). The research used complete randomized design (CRD) which is consisting of 12 treatments with 3 times repetitions. The parameters observed were time of callus formation, callus texture, callus color, callus pile height, callus surface area, callus weight, and callus performance. Data was analyzed by using ANOVA, and DMRT as follow up. The result showed that addition of plant growth regulators have effect on callus induction of shoot explant mangosteen (Garcinia mangostana L.). The best concentration and composition of media for induction of mangosteen callus is by giving sucrose 1,5 g / L + 2,4D 3 ppm because it was able to produce callus with the fastest time with a compact texture and color that is identical to green and white. The callus has 0.67a cm in height, 1.70a cm2 in width, and 0.51a g in weight. The callus performance is growing all over the surface.

Влияние инициальных сред и длительности холодовой предобработки на эффективность андрогенеза в культуре изолированных пыльников озимой пшеницы сорта Иркутская

Haploid technologies are based on the regeneration of gamete cells to form whole plants. The main advantage of haploid technologies is the ability to quickly in one generation obtain homozygous plants with the required parameters. Nevertheless, the use of these technologies today is limited, what is associated with insufficient knowledge of the physiological and biochemical features of the application of this method. In this regard, this work is devoted to the study of the features of androgenesis in vitro in the culture of isolated anthers of winter wheat cultivar Irkutskaya and the determination of the methodological features of obtaining intact plants. In the autumn-winter period, donor plants were grown at the artificial climate station SIPPB SB RAS. In the spring-summer period, donor plants were grown in the field at the experimental plots of the Zalarinsky agroecological station (Tungui village). The plant material was selected at the booting stage. Anthers were isolated from 6-10 spikelets of the middle third of the spike. The features of androgenesis were assessed by the frequency of formation of calli and embryo-like structures to the total number of anthers and the frequency of formation of the total number of seedlings and the number of green seedlings to the number of embryo-like structures and calli. It was found that the optimal medium for the induction of androgenesis in the anther culture of this common wheat cultivar is the modification of medium N6 containing 9% sucrose, with the addition of glycine (2 mg/L), 2.4-D (1 mg/L) and NAA (1 mg/L). The maximum yield of embryoid-like structures was observed in the case of low-temperature pretreatment of donor plants at + 4 °C for 6-12 days. At the same time, the most intensive calli formation in the culture of isolated anthers occurred after 3-6 days of donor plant cold pretreatment. Regeneration of green plants took place on 190-2Cu medium with the addition of growth regulators (2.4-D, 0.5 mg/L; kinetin, 0.5 mg/L) with obligatory cold pretreatment of androclinic structures at +4 °C for 3-7 days. Obtained results indicate that low-temperature exposure at different stages, from pretreatment of donor ears to exposure to androclinic structures, is a key factor for winter wheat that promotes androgenesis in the culture of isolated anthers and the successful formation of intact plants.

Effect of two cytokinins and a growth inhibitor on the in vitro tuberization of two genotypes of Solanum tuberosum L. cvs. Atlantic and Alpha

This investigation was carried out to evaluate the effect of two cytokinins: 6-benzilaminopurine (BAP) (6.5 mg l-1) and kinetin (K) (2.5 mg l-1), as well as the growth inhibitor abscisic acid (ABA) (1.0 mg l-1) on the in vitro tuberization capacity of two potato varieties: Atlantic and Alpha. The basal culture medium MS (1962) was used as a control. The responses were different between varieties. In cv. Atlantic, the analysis of the number (NM), weight (WM) and diameter (DM) of microtubers indicated that the addition of growth regulators did not affect induction and development of microtubers. However, when BAP was used, a non-significant increment of 41 % was observed in the number of the microtubers compared to the control treatment, from 2.6 to 4.4. The addition of cytokinins and ABA to the medium did not have a significant impact on the development of microtubers. In cv. Alpha the cytokinins used without ABA increased the number of microtubers, which were larger and heavier than those of the control treatment. In this variety, ABA significantly reduced the values of the NM, WM and DM variables. The exogenous action of cytokinins in the culture medium is likely to have caused an endogenous hormonal imbalance in the Atlantic and Alpha genotypes which interfered with their innate microtuberization ability, a result that was even more evident for cv. Alpha, which showed the need to continue optimizing protocols of genotype-specific systems in potato tissue culture to increase yield and seed quality.

Peculiarities of morphogenesis of the endangered species of wilow (Salix spp.) in vitro

Conservation and reproduction of rare genotypes of Salix L. species, in particular the blunt-leaved willow (Salix retusa L.) and Jacquin’s Willow (Salix alpina Scop.) that are listed in the Red Data Book of Ukraine in the status of rare and endangered species, is one of the urgent tasks of the present. The aim of research was to develop methods of introduction of S. retusa and S. alpina into in vitro culture for their mass reproduction and conservation. The plant material was cultivated on a culture medium prescribed by MS, WPM, and DKW with the addition of growth regulators according to the conventional method. Effective sterilization (over 80%) of explants of S. retusa and S. alpina was achieved by applying a stepwise method, which consisted of consistently maintaining them in solutions 0.1% HgCl2 and 1.0% AgNO3 for 5–6 min. Significant results in the regeneration of explants by activating the growth of available meristems in vitro were observed on MS with the addition of 0.25–0.5 mg/l 6-(Furfurylaminopurine, kinetin) and 2 g/l activated carbon. Our further research will serve as a base for developing microclonal propagation of S. retusa and S. alpina for their conservation and reproduction in vitro.

In Vitro Environmental Stresses for Enhancing Withanolides Production in Physalis angulata L.

Production of secondary metabolites through in vitro culture can be increased by providing environmental stress conditions. Therefore, the objective of this study was to determine the effect of in vitro environment stress by the addition of elicitor on the withanolides production of shoots culture of P. angulata. Two weeks old shoot culture of P. angulata was elicited in either MS medium + biotic elicitor (25, 75, and 125 mg/L chitosan) or abiotic (PEG 2.5, 3, and 5%) for two weeks. The control was MS0 medium without the addition of growth regulators or elicitor. The results of HPLC analysis showed that chitosan elicitor had better effect than PEG. Chitosan was more effective in increasing withanolide content than PEG with a little inhibition of growth. Elicitation with PEG slightly increased withanolide with more inhibiting shoot growth. This result shows that stress manipulation in the in vitro environment through elicitation is potential strategy to improve withanolides production from shoot culture of P. angulata.

HISTOLOGICAL ANALYSIS OF INDIRECT SOMATIC EMBRYOGENESIS INDUCED FROM ROOT EXPLATS OF OIL PALM (Elaeis guineensis Jacq)

ABSTRACT Oil palm is economically important as a crop with high oil production. Indirect somatic embryogenesis in oil palm requires a long time for callus induction and plant formation, and it is important to study the embryogenic potential of calli and the mechanisms of somatic embryogenesis. The aim of this study was to test different growth regulators and spermine in induction of embryogenic calli in root explants of oil palm (Elaeis guineensis Jacq). Apex root explants of approximately 0.5 cm were isolated from plants cultivated in vitro and inoculated in Y3 culture medium in the following treatments: A - without growth regulators; B - 1 mg.L-1 picloram (4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid); C - 1 mg.L-1 picloram and 2 mg.L-1 2ip (2-isopentenyladenine); D - 2 mg.L-1 2ip; E - 1 mg.L-1 picloram and 2 mg.L-1 BAP (6-benzylaminopurine); F - 2 mg.L-1 BAP; and G - 14.5 mg.L-1 spermine. After six months of culturing, the calli induced in the treatments were analyzed by light microscopy. The calli induced in the treatments with 1 mg.L-1picloram (B) and treatment with 14.5 mg.L-1spermine (G) exhibited embryogenic characteristics, small and isodiametric cells, forming agglomerates, besides a large amount of starch. Calli of the best treatment (Y3 com 1 mg.L-1 de picloram) were inoculated in Y3 culture medium without addition of growth regulators. After eight months, calli were once more analyzed under light microscopy. All the treatments showed callus formation, except for treatments D and A. Calli of treatment B exhibited cells with embryogenic characteristics that developed somatic embryos.