Combinations Of NAA Research Articles

The effects of plant growth regulators on shoot formation, regeneration and coloured callus production in Echinocereus cinerascens in vitro

Protocols were established for micropropagation and coloured callus production of Echinocereus cinerascens ,a cactus that is highly in demand for the horticultural industry. An efficient micropropagation system facilitates rapid shoot production of this threatened species. The present study investigates the effects of the plant growth regulators NAA and BAP on shoot and coloured callus induction. Shoots were produced from axillary buds and coloured callus from stem explants cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of NAA and BAP (0.5, 1.0, 1.5, 2.0 mg l -1 ) applied singly and in combinations. The medium that resulted in the highest mean number of shoots produced was MS supplemented with 2.0 mg l -1 NAA and 1.5 mg l -1 BAP. In general, media with combinations of BAP and NAA were more effective than NAA or BAP alone in inducing shoots. NAA alone resulted in significantly fewer shoots than BAP alone. Most stem explants produced coloured callus in all treatments. Green callus was induced in over 80 % of explants in all treatments in which the hormones were applied singly, while yellow callus was induced in 80-100 % of explants in half of the treatments with hormone combina- tions. In contrast, the occurrence of pink callus was less pre- valent than green and yellow callus. Pink callus was induced in 3-40 % of explants when treatments were added with single BAP or NAA and in 7-63 % of explants treated with combinations of BAP and NAA.

Establishment of an efficient and rapid method of multiple shoot regeneration and a comparative phenolics profile in in vitro and greenhouse-grown plants of psophocarpus tetragonolobus (L.) DC

An in vitro method of multiple shoot induction and plant regeneration in Psophocarpus tetragonolobus (L.) DC was developed. Cotyledons, hypocotyls, epicotyls, internodal and young seedling leaves were used as explants. MS media supplemented with various concentrations of either thidiazuron (TDZ) or N6-benzylaminopurine (BAP) along with NAA or IAA combinations were used to determine their influence on multiple shoot induction. MS media supplemented with TDZ induced direct shoot regeneration when epicotyls and internodal segments were used as explants. TDZ at 3 mg L−1 induced highest rate (89.2 ± 3.28%) of regeneration with (13.4 ± 2.04) shoots per explant. MS media supplemented with BAP in combination with NAA or IAA induced callus mediated regeneration when cotyledons and hypocotyls were used as explants. BAP (2.5 mg L−1) and IAA (0.2 mg L−1) induced highest rate (100 ± 2.66%) of regeneration with (23.2 ± 2.66) shoots per explant. Mature plants produced from regenerated shoots were transferred successfully to the greenhouse. In a comparative study, the phenolics contents of various parts of greenhouse-grown plants with that of in vitro-raised plants showed significant variations.

Optimization of genetic transformation of orchid (Dendrobium Sonia) mediated by Agrobacterium tumefaciens

Dendrobium Sonia is a popular orchid and used for many purposes in Vietnam. In this study, the transformation is set on protocorm like bodies (PLBs) mediated by Agrobacterium tumefaciens containing vector with GUS gene and kanamycin resistance gene. The main factors influencing the transformation including medium for regeneration and growth of PLBs, acetosyringone (AS) concentration, the co-cultivation time, Agrobacterium strain, bactericidal antibiotic concentrations and screening of transformed PLBs were surveyed. Results showed that MS medium supplemented with 1.0 mg/l NAA in combination with 0.5 mg/l or 0.3 mg/l BA was suitable for PLBs regeneration from stem tissue slices. The highest transformation efficiency (percentage of PLBs with GUS gene expression) was obtained when PLBs were infected and co-cultivated with Agrobacterium on the medium containing 100 µM AS for 3 days (with strain EHA105 and C58) or 4 days (with strain LBA4404). Medium containing 300 mg/l cefotaxime and 7-15 mg/l hygromycin was suitable for the disinfection and screening of transformed PLBs.

Effect of Nutrient Media and Phytohormones on<i> In Vitro</i> Establishment of <i>Bambusa balcooa.</i> Roxb.

Bambusa balcooa.Roxb. is one of the most commercially important bamboo species. Some varieties of bamboo are being grown in the company’s green house. However this species is very rare and it was not cultured until now due to lack of demand and maintenance problems. In this work B. balcooa ex-plants have been established and propagated by the axillary shoot bud proliferation method. Earlier several works have been done on this plant and a protocol has been. This work emphasizes on effects of phytohormones at different concentrations and combinations upon the novel propagules that develop from the bamboo explants on placing them in multiplication media. The propagules placed in media containing cytokinins BAP (2 mg/L) and Kinetin (1 mg/L) exhibited best results of linear as well as radial growth. The other propagules in media with combinations of BAP, Kinetin and NAA were found to be dried out and did not depict noticeable growth. Comparatively next best growth pattern to the former was observed in control media that lacked phytohormones. This experiment was helpful in estimating the

In vitro propagation of the bay laurel (Laurus nobilis.L) in Morocco

Laurus nobilis L. is among the species which are most threatened by massive degradation in Morocco. The multiplication by seed or by cuttings gives very low percentages of recovery that is insufficient to meet the demand of growing market. In vitro culture proves to be a tremendous asset to solve this problem. Our work has focused on the study of seed germination of this species and its multiplication from microcuttings. Finally, we studied the ac-climatization ability of the plantlets resulting from this germination. The study of the germination, via the further measurement of the length of the aerial part and the roots and the number of axillary buds for nine weeks, showed that the MS basal medium was more efficient than media 1/2M.S and WPM. Among the eight tested hormones, IAA yielded the best growth of the plantlets. Hormonal combination of NAA and kinetin resulted into a per-centage of the greatest success in reaching 67 % micropropagation. The study also revealed that the MS basal medium in the presence of the IAA plants can acclimate most easily in two types of substrates with improved development in the peat alone.

Effect of Nutrient Media and Phytohormones on<i> In Vitro</i> Establishment of <i>Bambusa balcooa.</i> Roxb.

Bambusa balcooa.Roxb. is one of the most commercially important bamboo species. Some varieties of bamboo are being grown in the company’s green house. However this species is very rare and it was not cultured until now due to lack of demand and maintenance problems. In this work B. balcooa ex-plants have been established and propagated by the axillary shoot bud proliferation method. Earlier several works have been done on this plant and a protocol has been. This work emphasizes on effects of phytohormones at different concentrations and combinations upon the novel propagules that develop from the bamboo explants on placing them in multiplication media. The propagules placed in media containing cytokinins BAP (2 mg/L) and Kinetin (1 mg/L) exhibited best results of linear as well as radial growth. The other propagules in media with combinations of BAP, Kinetin and NAA were found to be dried out and did not depict noticeable growth. Comparatively next best growth pattern to the former was observed in control media that lacked phytohormones. This experiment was helpful in estimating the

Agrobacterium-mediated transformation of embryogenic cell suspension cultures and plant regeneration in Lilium tenuifolium oriental × trumpet ‘Robina’

A method has been developed for embryogenic cell suspension cultures, plant regeneration and transformation of the important ornamental lily genotype (Lilium tenuifolium oriental × trumpet ‘Robina’). Bulb scales, filaments, ovaries and stem axis tissues were used as explants for callus induction in Murashige and Skoog (MS) medium with additions of growth regulators: picloram on its own, or in combination with 1-naphthaleneacetic acid (NAA), and thidiazuron (TDZ). The results show that the optimum medium for callus induction in bulb scale and filament tissue is MS + picloram 1.0 mg L−1, and for the ovary, it is MS + picloram 1.5 mg L−1. The stem axis had the highest rate (89.2 %) of callus induction with MS + NAA 2.2 mg L−1 + TDZ 0.1 mg L−1. The suspension cultures were established with the combination of NAA and TDZ with 2–5 mm cell clusters. These took a long time compared with suspension cultures established by picloram with 1–3 mm cell clusters. In three suspension cultures induced by picloram, the best callus from the point of view of proliferation and regeneration was derived from filaments. For plant regeneration, the growth rate of suspension cultures from the stem axis was higher than from the other three suspension culture induced by picloram. Vector pCAMBIA1301 with the β-glucuronidase (GUS) gene as reporter was transformed by Agrobacterium mediation into suspension cultures initiated from filament and stem axis material. After co-cultivation, the numbers of blue spots in material from the two sources were 26.8 ± 4.3 and 24.0 ± 4.7, respectively (difference not significant). Hygromycin-resistant callus was successfully regenerated into plantlets on plant growth regulator-free MS medium. Transgenic plants were also confirmed by the GUS histochemical assay, polymerase chain reaction.

Effect of the combination of NAA, kinetin and sucrose on the induction of somatic embryogenesis in lulo (Solanum quitoense Lam.)

Lulo is a species of great importance to the fruticulture of Colombia, but has significant phytosanitary problems that require an aggressive breeding program oriented toward the production of genotypes with tolerance to phytopathogens. These programs need to establish highly efficient mass plant propagation protocols, such as somatic embryogenesis. This study focused on research on the somatic embryogenesis of lulo using kinetin, naphthalene acetic acid-NAA (Plant Growth Regulators, PGRs), and different sucrose concentrations in a MS medium. Two lulo varieties, Solanum quitoense var. septentrionale and S. quitoense var. quitoense, and two explant types (hypocotyl and cotyledon) were used, incubated in dark conditions at 25±2°C. The highest production percentage of the embryos was obtained when 50 mM of NAA were added to the medium with sucrose (50.0 and 263.1 mM) for the two explant types used. In lulo with spines, the highest percentage of embryonic structures (50%) was observed with cotyledonary leaf explants and 50 mM of NAA ; while in the spineless lulo, the embryonic structures were observed in the same type of explant with 50 mM of NAA + 263.1 mM of sucrose (32%).

MORPHOLOGICAL AND HISTOLOGICAL EVENTS IN THE PRELIMINARY TISSUE CULTURE OF HAPLOID AND DIPLOID PELARGONIUM ZONALE VAR . 'KLEINER LIEBLING'

Pelargonium species are know and popular as the ornamental plants valued for their colorful, showy flowers and attractive leaf shape. Furthermore, some of these species are used in the pharmacy, aromatherapy, perfumery and cosmetic industry as well (Moyo et al. 2012). Most of approximately 280 species of the genus Pelargonium (fam. Geraniaceae) are native for South Africa (Mithila et al. 2001). Some of them such as P. zonale (L.) L'Her. ex Aiton were brought to Europe as early as in 1609. P. zonale var. ‘Kleiner Liebling’ is one of the pelargonium dwarf varieties with chromosomes number nine, and it is true haploid (monoploid) (Daker 1966), which makes it an interesting model plant.The aim of our study was the observation of in vitro culture of haploid and diploid plants of P. zonale var. ‘Kleiner Liebling’. It seems that this is the first attempt to establish an efficient protocol for regeneration in tissue culture conditions for these plants. These studies may serve as a start point for further studies of concerning plants at different ploidy levels.To obtain the plant regeneration, in the tissue culture conditions, we used explants both from mature haploid and diploid. Fragments of stems, parts of leaf petioles and leaf lamina were maintained on MS (Murashige and Skoog) basal media supplemented by several plants growth regulators. The first fodder contained 1 µM 2,4-D and 1 µM KIN; the second – 18,2 µM TDZ and 0,57 µM IAA; the third – 0,44 µM BAP and 0,54 µM NAA; and the fourth – 5,4 µM NAA and 0,44 µM BAP.The morphological and histological examinations showed the differences in morphogenetic response of haploids and diploids in applied tissue culture conditions. The response time for haploid and diploid P. zonale var. ‘Kleiner Liebling’ plants was also different. First of all, the callus formation on diploid stem explants was observed on eleventh day of culture on the medium with NAA and BAP combination. Two types of callus were observed on both haploid and diploid explants: endogenous and exogenous. The efficiency of callus production varied depending on type of explants used. The most frequent callus formation was found on fragments of stems and leaf blades (they responded in 100%), while leaf petioles were less efficient, 70% in diploids and 50 % for haploids.Organogenesis (mainly direct) was achieved in three types of explants; however, the response was different for haploids and diploids. Shoots were observed in most cases on haploid petiole leafs, whereas direct organogenesis in diploid explants was observed on stems and leaf blades.The optimal combination of plant hormones for the induction of adventitious shoots were TDZ and IAA (second fodder), while in the medium supplemented with 2,4‑D and KIN organogenesis was not recorded at all. Regenerated adventitious shoots were transferred to solid 1/2 MS medium, where their multiplication and successful development were observed.Direct rhizogenesis was induced only on stem explants. For haploids, roots were obtained on the medium containing TDZ with IAA and 2,4-D with KIN (the first and the second fodders), for diploids third and fourth media (supplemented with NAA and BAP in different concentrations) were suitable.The histological analysis confirmed the direct organogenesis and rhizogenesis, as well as heterogeneous origin of callus in diploid and haploid plants.In conclusion the crucial factor for organogenesis induction seems to be TDZ with combination with IAA application, what correlates with other experimental reports, where TDZ has been reported to induce adventitious shoot buds in number of plant species (Acharjee et al. 2012)In our experiments the somatic embryogenesis induction was not observed, although it is described in Pelargonium (Wojtania et al. 2004). However the SEM analysis showed the ECM (extracellular matrix) presence in diploid and haploid explants of leaf petioles, which may suggest the embryogenic potential of this experimental system. Future investigations are necessary for improving the culture protocols to obtain somatic embryos, plant regeneration and improvement of shoots formation.

English

The present study was initiated to optimize in vitro protocol for mass propagation of two commercial sugarcane clones (Co 449 and Co 678) grown in Ethiopia through shoot tip culture. Experiments on shoot multiplication and rooting were laid out in a completely randomized design with factorial treatment arrangements. Shoot tips were surface sterilized with 5% active chlorinated Berekina for 25 min and initiated on Murashige and Skoog (MS) medium supplemented with 2 mg L-1 6- benzylamino purine + 0.5 mg L-1 indole-3- butyric acid. For in vitro multiplication, aseptically initiated shoot tips were treated with different concentrations and combinations of BAP and Kin using either sucrose or table sugar in separate experiments. For root induction, regenerated shoots were transferred onto half MS medium supplied with 6% sucrose or table sugar and different concentrations and combinations of IBA and NAA. With regard to shoot multiplication, genotype Co449 showed maximum regeneration frequency of 80% with 7.87 ± 1.06 shoots per explants on MS medium with 3% sucrose and 2 mg L-1 BAP + 0.25 mg L-1 Kin. On the same carbon source, genotype Co678 showed the highest multiplication frequency of 90% with 9.10 ± 0.10 shoots per explant on medium supplied with 2 mg L-1 BAP + 0.5 mg L-1 Kin. On MS with 4% table sugar and 2 mg L-1 BAP + 0.25 mg L-1 Kin, 80% of the transferred explants of genotype Co449 produced multiple shoots with an average number of 7.61 ± 0.10 shoots per explant while genotype Co678 showed the highest regeneration frequency (86.67%) with mean shoots number per explant of 8.36 ± 0.04 on medium supplemented with 2 mg L-1 BAP + 0.5 mg L-1 Kin. Half MS + 6% sucrose + 2.5 mg L-1 IBA induced the highest rooting (86.67%) with an average root number per shoot of 16.53 ± 0.02 in Co449 cultures. In genotype Co678, ½ MS + 6% sucrose + 5 mg L-1 NAA induced the highest rooting response of 80% with an average root number per shoot of 13.17 ± 0.29. Equivalent rooting responses were recorded on half MS medium supplemented with 6% table sugar. On½ MS with 6 % table sugar, 2.5 mg L-1 IBA induced the highest rooting response (86.67%) and root number (15.93 ± 0.81) in genotype Co449 while 5 mg L-1 NAA gave the maximum (80%) rooting with average roots per shoot of 13.93 ± 0.81 in genotype Co678. Rooted shoots were transplanted in the green house for hardening and different survival rate was recorded.   Key words: Saccharum officinarum, multiplication, rooting, sucrose, table sugar, in vitro. &nbsp

EFFECT OF BA AND NAA ON IN VITRO MICROPROPAGATION OF TEA TREE

Induced explants of Melaleuca alternifolia by culturing on MS medium supplemented with different concentrations (0, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mg L-1) BA, with and without activated charcoal for 8 weeks. The result showed that the highest average new shoots (7.30 shoots/bunch) of tea tree were occurred in MS medium supplemented with 3 mg L-1 BA, but the average height was short and less than 1 cm. In this experiment, the activated charcoal did not enhance growth. When supplemented with NAA at different concentrations (0.5, 1.0, 1.5 and 2.0 mg L-1), with and without activated charcoal for 8 weeks. The results showed that MS medium supplemented with 2.0 mg L-1 NAA with activated charcoal induced the highest average number of roots whereas 7.3 roots, the highest number of leaf were at 21.2. When studied the combination of BA and NAA in MS medium on tissue of tea tree, the result showed that 0.5 mg/L BA combined with 2.0 mg L-1 NAA gave the highest average height (0.6 cm), leaves (11.9 leaves) and roots (2.3 roots).

Yabani Aspir'in (Carthamus persicus Wild) in vitro Çoğaltımında Farklı BAP-NAA Kombinasyonlarının Etkisi

Aspir, linoleik asit ve oleik asitce zengin; yenilebilir doymamış yağ asitlerinin elde edilmesinde kullanılması nedeniyle önemli bir endüstriyel bitkidir. Bu çalışmada aspirin in vitro’ da çoğaltımı amacıyla sürgün ucu eksplantları 0.25- 0.50- 1- 2 mg/L BAP ve 0.25- 0.50-1 mg/L NAA’ nın farklı kombinasyonlarını içeren, %3 sakkaroz ilave edilmiş ve % 0.8 agar ile katılaştırılmış MS ortamında kültüre alınmıştır. Araştırma sonuçları kallus oluşumu, rejenere sürgün yüzdesi, eksplant başına sürgün sayısı, sürgün uzunluğu, eksplant başına kök sayısı ve kök uzunluğu için BAP-NAA arasındaki kombinasyoların etkili olduğunu göstermiştir. Maksimum sürgün rejenerasyonu 0.50 mg/L BAP + 0.25 mg/L NAA içeren MS besin ortamında, maksimum kallus oluşumu ise 1 mg/L BAP + 1 mg/L NAA ve 2 mg/L BAP + 1mg/L NAA içeren MS besin ortamında kaydedilmiştir. Rejenere sürgünler 1 mg/L IBA içeren MS besin ortamında köklendirilmiştir.

Plantlet Regeneration from Seedling Explants of Sweet Gourd

To regenerate plantlets both directly and indirectly, different explants (cotyledon, hypocotyl, root tip and shoot tip) of sweet gourd (Cucurbita moschata) were cultured on medium supplemented with different concentrations and combinations of NAA (0.0, 0.2, 0.5 and 1.0 mg/L) and BAP (0.0, 0.5, 2.0 and 5.0 mg/L). Cotyledon explants performed best in callus induction. The combination of 5 mg/L BAP and 0.2 mg/L NAA produced the highest callus frequency in cotyledon (90.0%). The calli derived from cotyledon, hypocotyl and root tip were cultured in MS medium supplemented with different concentrations of kinetin, BAP and/or NAA for shoot induction. Regeneration via callus was achieved only from cotyledon calli at a frequency of 65.0% on 2 mg/L BAP and 0.2 mg/L NAA. Shoot tips cultured for direct regeneration, in the same media containing 1 mg/L BAP or 1 mg/L BAP + 0.2 mg/L NAA resulted in 100.0% shoot differentiation. The regenerated shoots rooted on MS medium with or without 0.1 mg/L NAA (100%).DOI: http://dx.doi.org/10.3329/pa.v18i2.18093 Progress. Agric. 18(2): 61 - 66, 2007

Effect of strigolactones and auxins on growth and metabolite content of Sutherlandia frutescens (L.) R. Br. microplants in vitro

The influence of strigolactones as hormones in plants is not fully characterised even though they are known to affect plant architecture, both above ground and in the roots. Using an in vitro system, the effects of the synthetic auxins 1-naphthalene acetic acid and indole-3-butyric acid (NAA and IBA) and synthetic strigolactones (GR24 and Nijmegen-1) were tested on microplant development of Sutherlandia frutescens, a leguminous medicinal plant native to South Africa. Considerable phytochemical variation in wild populations has led to the proposal of using micropropagation for this species. This will assist with domestication and provide plants with a more predictable chemistry for the phytopharmaceuticals industry. Nodal explants with an axillary bud were grown on Murashige and Skoog (Plant Physiol 15:473–497, 1962) medium [0.8 % (m/v) agar (pH 5.8), 3 % (m/v) sucrose and 0.1 g/L myo-inositol] supplemented with NAA, IBA, GR24 and Nijmegen-1, either singly or in combination. The amino acid profile and secondary metabolite pool was monitored using LC–MS-profiling. Treatment with NAA promoted mass shoot production, whilst a combination of NAA and Nijmegen-1 also positively influenced the accumulation of amino acids, flavonoids (sutherlandins) and terpenoids (sutherlandiosides) that S. frutescens produces. Since these compounds represent the presumed active compounds in this species and the biomarkers used in quality control assessment of S. frutescens tissues harvested for the pharmaceutical industry, this treatment holds promise for the commercial production of Sutherlandia extracts and herbal medications.

Callus induction and cell culture of castor (Ricinus communis L. CV. Shabje)

Context: Tissue culture is an important tool in plant biotechnology that allows for an increase in biomass or metabolite production by utilizing several techniques in callus or cell cultures. Ricin is a toxic protein that can be extracted from the castor bean as secondary metabolite. The ricin has been used experimentally in medicine to kill cancer cell. We developed callus and cell culture technique for the possible extraction of ricin from the cell suspension culture of Ricinus communis. Objectives: The present investigation aimed to develop the cell culture technique of R. communis L. cv. Shabje and tried to establish a protocol for cell suspension culture of castor for possible extraction of ricin from cell extract. Materials and Methods: The hypocotyl explants of R. communis L. cv. Shabje were used as experimental materials. Cultured on Murashige and Skoog medium supplemented with different concentrations and combinations of BAP, NAA, 2,4-D and IAA for callus induction. For cell culture, the media were used without agar with different concentrations and combinations of these hormones. Results: For callus induction the combination of BAP 2.0 mg/l + 0.5 mg/l NAA showed the best performance but in case of cell culture the combination of BAP 2.0 mg/l and 0.2 mg/l NAA showed the best result. Conclusion: The present investigation clearly established and demonstrated the method of obtaining cell suspension culture and important secondary metabolite ricin could be obtained from cell suspension culture of R. communis L. holding promises to explore cell culture industry for ricin production. DOI: http://dx.doi.org/10.3329/jbs.v20i0.17738 J. bio-sci. 20: 161-169, 2012

In vitro Root Culture : An Alternative Source of Bioactives in the Rare Aphrodisiac Herb Chlorophytum borivilianum Sant et Fern

Chlorophytum borivilianum is a rare, near-endemic high value root crop. The fasciculated roots of the plant have the potential to synthesize valuable bioactives having aphrodisiac, anti stress and immuno modulatory properties. The present investigation describes an alternative protocol for the development of a stable, fast growing, non-transformed root culture system in C. borivilianum and the simultaneous quantitative analysis of total steroidal saponins, amino acids and sugars. Liquid MS fortified with various concentrations and combinations of BA (0.44 - 8.8 µM), Kn (0.23 µM) and NAA (0.27 - 5.4 µM) was effectively used for the development of root culture system. High performance thin layer chromatography (HPTLC) was used for quantification and comparative analysis of bioactives from in vitro and in vivo grown tuberous roots. D. O. I. http://dx.doi.org/10.3329/ptcb.v23i2.17505 Plant Tissue Cult. & Biotech. 23(2): 133-146, 2013 (December)

Differentiation of shoot elements from the rachis of Secale cereale L.

<em>In vitro</em> culture of young <em>Secale cereale</em> spikes is described with special attention given to changes in development of the rachis. 7 mm explants were cultivated on a modified M u r a s h i g e and S k o o g (1962) medium (MS) supplemented with 2,4-D (dichlorophenoxyacetic acid), NAA (α-naph-thaleneacetic acid), IAA (β-indoleacetic acid), 2,4,5-T (trichlorophenoxyacetic acid), and ZEA (zeatin), KIN (kinetin), BAP (6-benzylaminopurine), IPA (izopentenyladenine or 2 iP) in numerous combinations and concentrations. Rachises differentiated branches with node construction in the presence of synthetic auxin. Rhizogenesis of explants was stimulated by 2,4-D and 2,4,5-T. 2,4,5-T + IPA appeared to be the best combination for callus regeneration. Many meristematic centres were found previously and combinations of NAA + KIN stimulated direct, whereas 2,4-D + ZEA indirect, differentiation of shoot elements. Development was observed from plantlets to flowering plants.

English

High frequency shoot regeneration from in vitro derived leaf explants of Wattakaka volubilis (L.f.) Stapf was achieved through callus mediated organogenesis. Organogenic calli were induced from 20 day old aseptic seedling explants on Murashige and Skoog medium fortified with various concentrations and combinations of plant growth regulators, benzylaminopurine (BAP), α-naphthaleneacetic acid (NAA), indole 3-butyric acid (IBA) and gibberellic acid (GA3). A mean of 8.6 shoots developed from organogenic callus induced from a 2 x 2 cm leaf explants on MS medium with 3% sucrose having 5.37µM NAA in combination with 2.22 µM BAP with 60% induction capacity. Further development of adventitious shoots could be achieved by sub culturing the callus to the same medium with 4.40 µM BAP and 0.288 µM GA3. Organogenesis could not be achieved from the calli of ex vitro derived leaf explants. The developed shoots were rooted on half-strength MS medium with 1% sucrose, 4.90 µM IBA and 0.93 µM kinetin at a frequency of 85%. Well rooted plantlets were then transplanted to vermiculite soil (3:1) mixture in polythene covered pots kept under culture room conditions. Approximately 60% plantlets survived and grew into whole plants. Key words: Adventitious shoots, caulogenesis, organogenic callus-histology.

Mass Propagation of an epiphytic orchid Acampe papillosa (Lindl.) through in vitro seed germination

A method of in vitro propagation of a medicinally important epiphytic orchid of NBU campus Acampe papillosa has been developed. Three different categories of media (1/2 MS, MS and Knudson C) were used for in vitro asymbiotic seed germination for conservation and commercial uses. Seed germination was observed in all the media without any growth supplement or additives although not efficiently. Seeds showed positive germination response in all the three media but the frequency and onset of germination, protocorm formation and associated morphogenic changes leading to plantlets establishment varied with the nature of additional growth stimulus. Different concentrations and combinations of auxin and cytokinins were used to enhance the germination and seedling development. The different concentration of coconut water (CW) was used as additive supplement in this study and 15% CW showed good germination within 22 days in KnC medium. Various concentrations and combinations of plant growth regulators BAP (1mg l-1 to 4 mg l-1), NAA (0.5 mg l-1 to 2 mg l-1) and IBA (0.5 mg l-1 to 2 mg l-1) were added into the three categories of media as growth stimulants. The induction of PLBs was observed in the absence of any exogenous supply of PGRs, but the addition of BAP (4 mg l-1) with KnC medium improved the shoot growth significantly and enhanced the PLBs development (95% efficiency) with highest shoot length (2.5 cm) after six months of culture. Minimum number of days was required for seedling development (148 days) when cultured into the KnC medium supplement with combinations of BAP (4 mg l-1) and IBA (2 mg l-1). Addition of small quantity of NAA (0.5 mg l-1to 2 mg l-1) was not improved the PLBs or seedling development in compared to BAP and IBA. Rooting was improved by addition of IBA (0.5 mg l-1 to 2 mg l-1) into the media. High rate of rooting efficiency was recorded when combinations of auxin and cytokinin was used. Frequency of rooting (95%) was studied in KnC medium containing BAP (4 mg l-1) and IBA (2 mg l-1). Seedling of 2.5 cm long with 3-4 leaves were transferred to earthen pot containing wood charcoal and coco peat for acclimatization under green house conditions. Hardened plantlets showed significantly high survival rate (81%) after seven months of transfer. Therefore, the present method of micro-propagation could be used successfully to propagate this important orchid for commercial production and conservation purposes.

Multiplicação in vitro de Caesalpinia pyramidalis (Leguminosae)

Caesalpinia pyramidalis is a species endemic to the Caatinga and known popularly as catingueira, which is widely used by local people, mainly for its timber and medicinal and fodder properties. This study investigated the effects of different types and concentrations of plant growth regulators on the in vitro multiplication of C. pyramidalis. In the first experiment, nodal segments were inoculated in media containing different combinations (0.0–8.0 µM) of BAP and NAA. In the second experiment, nodal segments wereinoculated in media containing different types (KIN, BAP and TDZ) and concentrations (0.0–16μM) of cytokinins. We used a WPM medium supplemented with 87.64 mM sucrose and solidified with 7.0 g L-1 agar. After 45 days, the highest number of shoots, leaf number, shoot length and dry mass of shoots were obtained when nodal segments were inoculated into a culture medium without plant growth regulators.