Nodular Callus Research Articles

Somatic embryogenesis and encapsulation of immature bulblets of an ornamental species, grape hyacinths (Muscari armeniacum Leichtlin ex Baker)

An efficient in vitro regeneration system was developed for the ornamental Muscari armeniacum on Linsmaier and Skoog basal medium (LS) supplemented with benzyladenine (BA) at varying concentrations (0.5, 1.0, or 2.0 mg/L) alone or in combination with 0.5 mg/L alpha-naphthalene acetic acid (NAA). The highest mean number of direct somatic embryo formations was observed on LS medium containing 2.0 mg/L BA and 0.5 mg/L NAA, with a mean of 7.9 somatic embryos per explant after 10 weeks of culture. Green nodular calli induced on LS medium containing 5.0 mg/L BA alone or in combination with 0.5 mg/L NAA were transferred to LS medium supplemented with or without 0.5 mg/L gibberellic acid (GA3) for 8 weeks, producing 23.3 immature bulblets. Immature bulblets produced in vitro were either embedded in a sodium alginate matrix for the encapsulation process or were transferred directly to LS medium supplemented with or without GA3 at 0.5 or 1.0 mg/L for growth and development for 6 weeks. Encapsulated bulblets were then stored at 4 °C in darkness for 10 weeks and almost all encapsulated bulblets retained their viability and resumed their growth under nonaxenic greenhouse conditions.

Histological and biochemical parameters of Crocus sativus during in vitro root and shoot organogenesis

Content of malondialdehyde (MDA), proline, phenolics, and saccharides was analyzed during different developmental stages of in vitro root and shoot organogenesis in saffron. The highest content of MDA, proline, and phenolics was detected in nodular calli. Significant changes were also found in the content of polysaccharides, soluble saccharides, oligosaccharides, and reducing saccharides during developmental stages. Histological investigation of nodular calli showed meristematic zones with small and densely stained cells situated at peripheral zones of calli. The meristematic zones surrounded some vascular areas from which de novo organs originated. The parenchymatic cells of inner zones of calli converted to procambium cells that produced vascular tissues.

Indirect Organogenesis and Histological Analysis of Garcinia mangostana L.

Abstract: The improvement of Garcinia mangostana L. through biotechnology approach needed an efficient plant regeneration system. The objective of this research was development of indirect organogenesis protocol for mangosteen such as effect of plant growth regulator for induction of nodular calli, plant regeneration and histological analysis of nodular calli and shoot bud. The treatments for induction nodular calli were concentration of BAP 2.2 and 4.4 uM while concentration of TDZ (thidiazuron) 1.14, 2.27 and 4.54 p.M. Combination of BAP and TDZ concentration used as treatments. The medium of shoot proliferation was Woody Plant Medium (WPM) with concentration of BAP (0.0; 1.1; 2.2; 3.3; 4.4) uM as treatments. All experiments were arranged completely randomized design and analyzed data using F-test and the means among each treatment was separately by Duncan's Multiple Range Test (DMRT). A result showed that combination of 2.22 and 2.27 itM TDZ on MS medium produce nodular calli basal medium WPM with 2.2 itM BAP concentration indicated the highest percentage of nodular callus formed shoot was 34.7%, average of numbers shoot per nodular calli was 7.8 shoots, average of time formed shoot was 94.5 days and number of shoot length 1-5 mm was 11.06, shoot length 6-10 mm was 2.61 and shoot length >10 mm was 0.61. The highly efficient protocol of indirect organogenesis suggested in this study can be used to mutation breeding methods and propagation of Garcinia mangostana L.

Fitorreguladores e posição de explantes foliares na indução à calogênese em cerejeira-do-mato

O presente trabalho objetivou avaliar o efeito de diferentes formas de inoculação, no meio nutritivo, de explantes foliares de Eugenia involucrata DC., uma espécie florestal nativa com diversas potencialidades econômicas. Foram avaliadas as posições abaxial, adaxial, com e sem cortes no limbo foliar. Foi utilizado o meio de cultura MS acrescido de 10 µM de ANA isolado ou das combinações, em µM, de 2,4-D e BAP: 5-5 e 5-10. A posição dos explantes afeta a calogênese e a organogênese em segmentos foliares de E. involucrata, sendo mais adequada a abaxial sem cortes na região do limbo. A associação dos reguladores de crescimento 2,4-D + BAP na concentração de 5-10 µM foi mais promissora para a obtenção de calos, especialmente os nodulares, putativos à embriogênese somática.

EMBRYOGENIC POTENTIAL OF CALLUS DERIVED FROM AN ADULT GIANT BAMBOO (Dendrocalamus giganteus Wall. ex Munro)

Dendrocalamus giganteus Wall.ex Munro (giant bamboo), the largest of the bamboos in SriLanka, is recommended for expanded cultivation. Natural means of propagation by vegetative orsexual methods is limited in this species. In the absence of a true breeding system, novelbiotechnological methods offer a para sexual alternative for genetic improvement. This wouldrequire de novo plant regeneration via callus, cell suspensions or protoplasts. With this objectivein view, callus was induced from axillary shoots, spikelets and roots that formed in vitro in acontinuously proliferating system of axillary shoots derived from a 70 year old field clump of D.giganteus. The callus proliferated in MS medium containing 4% sucrose, 2,4-D (7.5 rngl) andNAA (3 mgl) to form nodular callus that showed a potential for plantlet re~eneration. Thefriable callus readily formed cell suspensions in the presence of 2,4-D (7.5 mgl' ) along or withNAA (3 mgl), These cells showed organized development to form structures that resembledpro-embryos. Organogenesis/embryogenesis is novel from long established adults although it ismore common in juveniles. This report signifies the potential of a 70-year-old adult bamboo for plantlet regeneration.

Callus induction and plant regeneration of paederia foetida L., a widely used medicinal vine in Bangladesh

An efficient protocol was established for in vitro plant regeneration of Paederia foetada L. (Family. Rubiaceae), a widely used medicinal vine of Bangladesh through callus culture in using nodal segment. Yellowish green nodular callus was observed from nodal segments on MS basal medium supplemented with 1.5 mg/L BAP + 0.5 mg/L NAA within three weeks. Large number of shoots (14.4±1.29) were obtained when the callus was sub cultured on MS medium with 0.5 mg/L BAP. In vitro raised shoots rooted on half strength MS medium with 0.5 mg/L IBA. The survival rate of plantlets was found to be 85%. Regenerated plants were morphologically uniform having normal leaf shape and growth. Bangladesh J. Sci. Ind. Res. 47(4), 373-378, 2012 DOI: http://dx.doi.org/10.3329/bjsir.v47i4.14066

Highly efficient system for plant regeneration from leaf and stem explants in Dahlia

Protocol of plant regeneration from leaf and stem explants was established in dahlia (Dahlia×pinnata) ‘Yamatohime’ in in vitro culture. Nodular calli were successfully induced from leaf and stem explants on 30 g l−1 sucrosecontaining MS medium supplemented singly with one of the cytokinins, 6-benzyladenine (BA), zeatin or thidiazuron (TDZ) and the highest frequency of nodular callus induction was obtained on medium containing 10 mg l−1 TDZ. The nodular callus induced on this medium subsequently produced a tissue with numerous shoot primordial structures called ‘mass of shoot primordia’ (MSP), which was maintained at least for more than 2 years on the same medium. Shoot induction occurred after transfer the MSP onto hormone-free MS medium, in which replacement of sucrose by maltose was necessary. The shoots excised from the MSP were rooted on hormone-free MS medium and successfully grown under greenhouse conditions. The regenerated plants showed the same morphological characteristics to those of the control plants and flowered normally.

Callus Induction and Plant Regeneration from Immature Embryos of Zelkova sinica Schneid.

Zelkova sinica Schneid. is a popular landscape plant in China because of its wide adaptation, strong disease resistance, large crown, and beautiful fall color. Immature embryos from Z. sinica seeds were cultured on woody plant medium (WPM) supplemented with 4.5 μM 6-Benzylaminopurine (BA) and 5.4 μM α-naphthaleneacetic acid (NAA) to induce callus, and 60% of immature embryos formed callus. The cream-white, friable, nodular callus with proembryogenic structures was then cultured on WPM containing 5.4 μM NAA in combination with 9.0 or 11.2 μM BA to regenerate shoots; approximately five shoots per explant were induced on 70% callus. Shoots were rooted on WPM containing 0.5 μM indole-3-butyric acid (IBA), on which 62.3% shoots developed roots with an average of 4.2 roots per shoot at 4 weeks. The regenerated plantlets were acclimatized and transplanted into the field. This protocol could be used for mass production for field plantation, genetic improvement, and germplasm exchange of Z. sinica.

In vitro development of embryogenic calli and embryogenic stages in suspension cultures of mangosteen (Garcinia mangostana L.)

In this study, the potential of uncoated and coated mangosteen seed explants in forming embryogenic callus were examined in the basal Linsmaier and Skoog medium supplemented with different auxins at various concentrations. Among the highest percentage of callus response (93.3%) was obtained when uncoated seed explants were cultured in basal LS medium containing 8 mg/L 2,4-D. Combining of cytokinins and 2,4-D to improve embryogenicity of calli showed that among the highest percentage of callus response (80%) and the lowest percentage of callus browning (53.53%) with yellowish and compact nodular calli were obtained on MS medum supplemented with 8 mg/L 2,4-D and 0.1 mg/L BAP. Addition of glutamine into MS medium containing 8 and 0.1 mg/L BAP performed that glutamine did not increase the growth of calli, however texture (friable) and color callus (yellowish) was improved. The growth and multiplication of cells in suspension cultures showed that the cells were able to divide and proliferate even though cultured in half strength MS liquid medium without 2,4-D. After six months of culture, the heart embryogenic stage was obtained only on medium supplemented with 1 mg/L BAP. The globular and torpedo embryogenic stages were obtained on media supplemented with 1, 3 and 9 mg/L TDZ after five months of culture. Key words: Mangosteen, embryogenic callus, suspension culture, in vitro.

Rapid and stable in vitro regeneration of plants through callus morphogenesis in two varieties of Mucuna pruriens L. – an anti Parkinson’s drug yielding plant

Mucuna pruriens L. is an important medicinal plant reported to contain L-3,4-dihydroxy phenylalanine (L-DOPA). The present report describes a simple protocol for plant regeneration through callus morphogenesis in four strains belonging to two different varieties of Mucuna pruriens. Friable, soft and nodular callus was induced from nodal and internodal segments of in vitro-grown seedlings on modified Murashige and Skoog’s (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP). For regeneration of shoots, the callus tissues were transferred to medium supplemented with various levels of cytokinin. Highest shoot regeneration from the callus tissue was achieved in modified MS medium fortified with BAP at 1.33 μM level. The regenerated shoots were rooted in vitro in half-strength of liquid MS medium supplemented with various levels of NAA. The regenerates were acclimatized for 2–3 weeks and showed about 80 % survival rate after transferring to the field. Cytological analysis revealed chromosome number stability of all the regenerates (2n = 22) with complete absence of aneuploidy. Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) analyses of total genomic DNA supported stability at the molecular level among all the clones of these strains when compared with their mother plants.

Reproducible and expedient rice regeneration system using in vitro grown plants

Inevitable prerequisite for expedient regeneration in rice is the selection of totipotent explant and developing an apposite combination of growth hormones. Here, we reported a reproducible regeneration protocol in which basal segments of the stem of the in vitro grown rice plants were used as ex-plant. Using the protocol, callus was developed from the exposed cells of root segments and cortical tissues of basal part of the stem. Various levels of 2,4-dichlorophenoxyacetic acid (2,4-D) were used, where 1 mg/L was found as best level for callus induction. Further, 25 combinations of kinetin and naphthalene acetic acid (NAA) were developed to investigate the regeneration response of the calli. The root-derived calli did not respond to any combination at all, whereas the nodular calli derived from the stem segments responded variably to kinetin and NAA combinations in the Murashige and Skoog (MS) medium from base to top. Higher kinetin to NAA ratios promoted embryogenesis, whereas lower ratios exhibited rhizogenesis. A combination of 3 mg/L kinetin and 1 mg/L NAA was established to be the best combination for plant regeneration through embryogenesis in rice from in vitro grown plants. Plants regenerated in vitro were successfully acclimatized in the pots, where they exhibited phenotypically indistinguishable normal growth when compared with plants developed from seed, hence the developed regeneration system for rice in these studies may be treated as one of the best strategies to in vitro clonal propagation and purification, ahead of seasonal growth of plants in the field or green house.

In Vitro Regeneration of Saccharum edule From Immature Inflorescence

Saccharum edule is grown in Pacific islands for its edible, aborted inflorescence. This species is not amenable for conventional hybridization programme for improvement of sugarcane as it does not produce normal flowers. For in vitro multiplication of this species, immature inflorescence slices of 1 mm thick were inoculated on modified MS medium supplemented with 1 mg l−1 NAA + 2,4-D (5–50 mg l−1). Compact and nodular calli were observed in medium supplemented with 40 mg l−1 2,4-D. This calli were sub-cultured on medium supplemented with 1 mg l−1 NAA + BAP (2–10 mg l−1), good regeneration (327 shoots/Explant) were obtained. The regenerated plants were planted in field after rooting and hardening. The field grown plants were morphologically similar to the parental clones with similar aborted inflorescence.

Regeneration of pineapple (Ananas comosus L.) plant through somatic embryogenesis

An efficient protocol for a complete plant regeneration by somatic embryogenesis was developed with Smooth Cayenne pineapple (Ananas comosus L.). Previous works showed that this species is responsive to somatic embryogenesis. In the present work the influence of components of culture medium in the induction, development and conversion of somatic embryos was investigate in order to establish a somatic embryogenesis protocol. Nodular callus (83.67%) was initiated from leaf explants of young plants on CIM3 medium. The highest frequency (37.6%) of embryogenic callus induction was obtained from 4-week-old calluses on EIM3 medium supplemented with 3.0 mg/l picloram. The highly organized callus induction and the development of somatic embryos were achieved after the transfer of callus clumps onto EIM3 medium containing 1.0 mg/l BAP + 0.1 mg/l NAA. The frequency of somatic embryo formation was of 39.5 ± 2.45 embryos per callus. Up to 97% of the somatic embryos were converted into complete plants within 4 week on MSB medium with 1.0 mg/l BAP + 0.05 mg/l GA3 + 500 mg/l glutamine. The continuation of the elongation of the shoots occurred on this medium). Shoots obtained from all the above methods were rooted in MSB medium with activated charcoal. Complete plantlets were transferred onto specially made polyethylene bags containing soil mixture and transferred to the greenhouse. Survival rate of the plantlets under ex vitro conditions was 98% and maximum average number of plantlets (80 ± 0.6). The well-developed plantlets were transferred to an open field where the plants produced normal fruits.

Genetic transformation of Indian isolate of Lemna minor mediated by Agrobacterium tumefaciens and recovery of transgenic plants

Transgenic plants of an Indian isolate of Lemna minor have been developed for the first time using Agrobacterium tumefaciens and hard nodular cell masses 'nodular calli' developed on the BAP - pretreated daughter frond explants in B5 medium containing sucrose (1.0%) with 2,4-D (5.0μM) and 2-iP (50.0μM) or 2,4-D (50.0μM) and TDZ (5.0μM) under light conditions. These calli were co-cultured with A. tumefaciens strain EHA105 harboring a binary vector that contained genes for β-glucuronidase with intron and neomycin phosphortransferase. Transformed cells selected on kanamycin selection medium were regenerated into fronds whose transgenic nature was confirmed by histochemical assay for GUS activity, PCR analysis and Southern hybridization. The frequency of transformation obtained was 3.8% and a period of 11-13weeks was required from initiation of cultures from explants to fully grown transgenic fronds. The pretreatment of daughter fronds with BAP, use of non-ionic surfactant, presence of acetosyringone in co-cultivation medium, co-culture duration of 3d and 16h photoperiod during culture were found crucial for callus induction, frond regeneration and transformation of L. minor. This transformation system can be used for the production of pharmaceutically important protein and in bioremediation.

Somatic embryogenesis and plantlet regeneration from immature anthers of Opuntia ficus-indica

SummaryAn in vitro protocol has been developed for callus induction, somatic embryogenesis, and plant regeneration in the Barbary fig (Opuntia ficus-indica) cultivars ‘Gialla’ and ‘Moore’. Immature anthers cultivated in the dark on induction medium which consisted of Chée and Pool medium containing 2.0 mg l–1 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.5 mg l–1 thidiazuron (TDZ), produced primary nodular and greenish calli within 6 – 8 weeks. Periodic transfer of nodular calli on induction medium in the dark induced the formation of compact embryogenic masses after 4 – 5 months of cultivation, with induction rates of 58.97 ± 1.10% and 61.15 ± 1.16% for ‘Moore’ and ‘Gialla’, respectively. Various distinct stages were observed during the development of somatic embryos on induction medium, including pro-embryogenic, globular, heart-, torpedo-, and cotyledonary-shaped somatic embryos, as well as secondary somatic embryos. Further development of somatic embryos was accomplished using half-strength Murashige and Skoog medium containing 1% (w/v) activated charcoal under a 16-h photoperiod. Bipolarity of the somatic embryos was confirmed by environmental scanning electron microscopy. Regenerated plantlets with well-developed cladodes and roots were transferred to a greenhouse and acclimatised successfully.

High frequency in vitro propagation of Trichopus zeylanicus subsp. travancoricus using branch–petiole explants

Trichopus zeylanicus subsp. travancoricus (known as Arogyapacha), an endangered ethnomedicinal plant of the Western Ghats of South India, serves as the major source of the commercial drug Jeevani. The present study established a long-term high frequency in vitro propagation protocol for Arogyapacha. Callus obtained from the branch–petiole explants cultured on Murashige and Skoog (MS) medium with 4.5 μM 2,4-dichlorophenoxyacetic acid upon subculture to medium with different concentrations of 6-benzyladenine (BA) either alone or in combination with an auxin favoured shoot morphogenesis. Medium with 13.3 μM BA alone facilitated high frequency shoot bud (mean of 93.2) formation. Medium with lower concentrations of BA (4.4, 6.6 and 8.8 μM) alone or in combination with lower concentration of α-naphthaleneacetic acid (NAA) or indole-3-butyric acid (IBA) favoured better shoot growth than 13.3 μM BA containing medium, but with reduced number of shoot buds. Subsequent cultures on medium with lower concentrations of BA and also on MS basal media facilitated shoot formation as well as growth of shoots. The shoot regeneration potential showed no decline up to 5 years. Culture of the in vitro-derived whole branch–leaf explants on MS basal medium developed shoots directly from the node. On medium with 19.6 μM IBA, the whole branch–leaf explants induced nodular callus from the node, which developed shoots later. Subsequent cultures on medium with BA exhibited high frequency shoot formation. The transfer of shoots after 10–15 days culture on half-strength MS medium containing 2.7 μM NAA to half-strength basal medium induced a mean of 11.3 roots. Field survival of plantlets relied on the soil mix: a 1:4 ratio of sand and red-soil exhibited the highest plantlets survival (86.6%). RAPD profile of the source plant and plants regenerated from calli after 4 years showed no polymorphism. The established plantlets with morpho-floral features similar to that of the source plants flowered normally and set fruits.

A micropropagation protocol for Cassia angustifolia Vahl. from root explants

The aim of this study was to develop a new micropropagation system for Cassia angustifolia Vahl., an important medicinal legume using root explant as starting material. Root explants taken from 30-day-old aseptic seedlings were cultured on Murashige and Skoog (MS) medium supplemented with different plant growth regulators: 6-benzyladenine (BA), kinetin (Kn), and thidiazuron (TDZ). Organogenic nodular calli obtained on MS + TDZ (1.0 μM) were transferred to shoot regeneration medium supplemented with different cytokinins (BA, Kn or TDZ) either alone or in combination with auxin:indole-3-acetic acid or α-naphthalene acetic acid. Maximum shoot regeneration frequency (90%) was obtained on MS + BA (2.5 μM) + NAA (0.6 μM) wherein a maximum of 42.76 ± 1.47 shoot buds per explant were induced with a maximum conversion rate of 35.63 ± 0.75 shoots per explant and average shoot length of 5.43 ± 0.20 cm. Elongated microshoots were successfully rooted under ex vitro conditions by pulse treatment in 200 μM of indole-3-butyric acid for half an hour. Microshoots were rooted, acclimatized and hardened off simultaneously in sterilized soilrite inside the growth room and then established in pots containing sterilized soil and manure (1:1) and grown under greenhouse condition with 90% survival rate. The histological sections at different developmental stages of shoot buds revealed the organization of nodular meristematic zone leading to the orientation and differentiation of shoot buds in large number and thereafter conversion into healthy shoots.

Somatic embryogenesis and plant regeneration from callus cultures of Cleome rosea Vahl

This paper describes a protocol for the efficient vegetative propagation of Cleome rosea by somatic embryogenesis. Leaf and stem explants from nursery-grown seedlings of C. rosea were cultivated on Murashige and Skoog (MS) medium supplemented with indole-3-acetic acid (IAA), a -naphthaleneacetic acid (NAA), 4-amino-3,5,6-trichloropicolinic acid (picloram) or 2,4-dichlorophenoxyacetic acid (2,4-D). Nodular calli were produced from both explant types in the presence of 4.5 and 9.0 µM 2,4-D. Embryo development and maturation were achieved when calli from stem explants were transferred to media containing a ten-fold reduction of 2,4-D concentration initially used (0.45 and 0.90 µM). Leaf-derived calli did not form embryos with the same treatments. The highest frequency of embryogenic callus formation (85%) and number of embryo per callus (13.45 ± 2.8) were achieved during the first subculture on medium supplemented with 0.90 µM 2,4-D. Embryo conversion into plantlets was achieved following transfer to growth regulator-free MS medium solidified with 2 g.L-1 phytagel. An acclimatization rate of 53% was found three months after transfer to ex vitro conditions and the recovered plants presented a normal phenotypic aspect.

Efficient and rapid plant regeneration of oil palm zygotic embryos cv. ‘Tenera’ through somatic embryogenesis

An efficient and rapid plant regeneration system through somatic embryogenesis was developed using 13-week-old zygotic embryos of oil palm (Elaeis guineensis Jacq.) cv. ‘Tenera’. Zygotic embryos were cultured on MS and N6 media supplemented with 2.0 mg L−1 picloram, 2,4-D and dicamba. The highest embryogenic callus formation (32%) was observed on N6 medium with 2,4-D after 3 month culture on callus induction medium. Somatic embryos were continuously formed from nodular calli on embryo maturation medium [N6 + 0.1 mg L−1 2,4-D, 0.16 g L−1 putrescine, 0.5 g L−1 casein amino acids and 2.0 g L−1 activated charcoal(AC)] for 3–5 months. Histological analysis confirmed that embryo development occurred via somatic embryogenesis. For plant regeneration, modified N6 medium (MN6) with AC (0.5 g L−1) without growth regulators, induced both shoot and root formation simultaneously with the highest regeneration rate of 56%. This combined shoot and root induction protocol shortened the culture time to 9–12 months. Furthermore, after acclimatization, more than 85% of transferred plants from our protocol developed successfully in the soil.

Macro elements in inoculation and co-cultivation medium strongly affect the efficiency of Agrobacterium-mediated transformation in Lilium

A highly efficient Agrobacterium-mediated transformation system for Lilium × formolongi was established by modifying the medium used for inoculation and co-cultivation. Meristematic nodular calli of Lilium were inoculated with an overnight culture of A. tumefaciens strain EHA101 containing the plasmid pIG121-Hm harboring an intron-containing β-glucuronidase (GUS), hygromycin phosphotransferase, and neomycin phosphotransferase II genes. The effects of ten different types of media and carbohydrates (sucrose, d-glucose, and l-arabinose) in both inoculation and co-cultivation media were evaluated. Interestingly, a dramatic increase in the frequency of transformation (25.4%) was observed when Murashige and Skoog (MS) medium containing sucrose and lacking KH2PO4, NH4NO3, KNO3, and CaCl2 was used. Hygromycin-resistant transgenic calli were obtained only in medium supplemented with sucrose. The effects of this modified medium were also investigated for Lilium cultivars ‘Acapulco’, ‘Casa Blanca’, and ‘Red Ruby’. The highest frequency of transformation (23.3%) was obtained for cv. Acapulco. Hygromycin-resistant calli were successfully regenerated into plantlets on plant growth regulator-free MS medium. Transgenic plants were confirmed by GUS histochemical assay, polymerase chain reaction (PCR), and Southern blot analyses.