Embryogenesis and Differentiation in Nigella sativa Leaf Callus in vitro

Embryogenesis occurred in Nigella sativa L. (Fam. Ranunculaceae) leaf callus tissue when coconut milk was replaced from the Murashige and Skoog's (MS) medium by casein hydrolysate. On MS + IAA (0.5 mg/l) + casein hydrolysate (100 and 500 mg/l) medium, tissue gained a capacity of growing embryoids for a pro-longed culture period. At a concentration of 1000 mg/l casein hydrolysate suppressed the differentiating capacity after the fifth subculture. 2.4-D and kinetin had inhibitory effects on morphogenesis. Histology of the differentiated tissue revealed that the origin of roots, shoot buds and leaves were from groups of meristematic cells whereas embryoids were initiated by the repeated division of a single cell.     

Differentiation in Callus Cultures of Leaf of Two Species of Trigonella

Callusing of leaf segment of Trigonella corniculata L. and Trigonelta foenum graecum L. was observed on Murashige and Skoog's (MS) medium and White's (W) medium supplemented with naphthalene acetic acid (NAA; 0.5 mg/1) and coconut milk (15% v/v). Root, shoot and development of isolated leaf occurred in the calli of both the species on MS medium containing NAA amd coconut milk. Omission of coconut milk with subsequent addition of casein hydrolysale (CH) increased the number of differentiated organs per callus. In both the materials higher differentiation rate was observed at low irradiance than at high irradiance or in complete darkness. Initially the cells were diploid but at the 5th passage the calli became mixoploid wilh predominantly diploid cell populations. The differentiated organs were diploid.     

In vitro Studies on Pinus

Hypocotyl tissue from Pinus gerardiana was established in culture on White's basal medium supplemented with 2 % sucrose, 10% (v/v) coconut milk, 500 mg/l casein hydrolysate and 1 mg/1 2,4-dichlorophenoxyacetic acid (2,4-D). Various organic and inorganic nutrients were studied in order to determine the specific nutritional requirements of the tissue in vitro. Sucrose, glucose and maltose were equally effective as fixed carbon sources. The inorganic nutrient combination of White's medium was found to be better than that of Murashige and Skoog's medium. White's modified basal medium supplemented with coconut milk, casein hydrolysate and 2,4-D was the most successful nutrient combination. Glutamine was as effective as casein hydrolysate in promoting growth of the tissue.     

Plant regeneration through somatic embryogenesis from mature seed and young inflorescence of wild rice (Oryza perennis Moench)

Somatic embryogenesis in the wild rice species (Oryza perennis) was induced from cultured mature seeds and young inflorescences. Murashige and Skoog's (MS) medium supplemented with 2 mg/l 2,4-D and 0.2 mg/l BAP was used for induction of a compact, white nodular callus and somatic embryos. Plant regeneration occurred with the tranfer of the nodular callus to MS basal medium containing 0.5 mg/l IAA, 0.5 mg/l NAA, 4 mg/l BAP and 500 mg/l casein hydrolysate. The embryogenic nature of the callus from both explants was maintained over 10 subcultures for about 12 months. Plant regeneration with respect to the number of calli plated from the 6th to 10th passage varied from 80% to 60% for young inflorescence derived callus and from 75% to 69.8% for seed-derived callus.Abbreviations MS Murashige and Skoog medium - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA naphthalene acetic acid - CH casein hydrolysate     

Nutrient Requirements of Vigna sinensis Callus Cultures

Nutrient requirements of callus tissues previously obtained from Vigna sinensis hypocotyl segments were investigated on modified White's (Miller and Skoog) and Murashige and Skoog's media containing 1 mg/1 of each of 2,4-D and kinetin, 2% sucrose and 1% agar. Yields of tissues on the latter medium were 3 times more than the former. After studying various organic and inorganic nutrients, two synthetic media have been developed for callus cultures. The above modified White's medium further supplemented with 2% sucrose, and 500 mg/1 of urea or asparagine was suitable for a compact callus with a dry-to-fresh-weight ratio around 8% and on this the tissues grew 15- to 17-fold in 5 weeks. Similarly the Murashige and Skoog's medium further enriched with 2% sucrose, 400 mg/1 of urea or a mixture of aspartic acid (750 mg/1), glutamic acid (250 mg/1), asparagine (250 mg/1) and arginine (250 mg/1), was apt for a friable callus with a dry to fresh weight ratio of 3 to 4% and it promoted a 45- to 50-fold increase in growth in 5 weeks.     

Rapid mass propagation of Chrysanthemum morifolium by callus derived from stem and leaf explants

A procedure for rapid multiplication of Chrysanthemum morifolium RAMAT cv. Birbal Sahni using leaf callus and stem (nodal/internodal) callus as well as node and apical shoots has been developed. Murashige and Skoog's medium (1962) supplemented with 2mg/1 2,4-D yielded good green calli from both leaf and stem segments within 2 weeks. About 1 cm × 1 cm callus regenerated 2–3 shoots after 3 weeks on MS solid medium supplemented with 0.1 mg/l IAA and 0.2 mg/l BAP. Each of the regenerated shoots when transferred to the same shooting medium without agar yielded about 150 new shoots, which in turn regenerated roots after another week in MS half strength or modified White's media (Rangaswamy, 1961). It has been estimated that about 10¹⁴ plantlets could be produced in a year from one expiant following the proposed protocol.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog's (1962) medium     

The Effects of Agitated Liquid Medium on in vitro Cultures of Hevea brasiliensis

The initiation and prolonged growth of callus, from stem explants of young plants of Hevea brasilienies on solid medium yielded a heterogeneous callus, with areas which are the result of compact growth interspersed with brown necrotic tissue and soft white tissue formations. Subculturing this callus (O callus) to agitated liquid medium and returning it to solid medium resulted in the production of a homogeneous friable and rapidly growing callus (Rl callus) The two established lines O and Rl have remained stable over one year in culture and differ in gross morphology, anatomy, growth and auxin content. Both were maintained on Murashige and Skoog's medium, with 2 mg/1 2,4-D and 0.5 mg/I kinetin. R 1 but not O showed enhanced growth at the lower 2,4-D level of 0.2 mg/l: both lines failed to continue growing when 2,4-D was omitted. It is suggested that the changes resulting from subculture in agitated liquid medium are related to those undergone by callus cultures which become habituated. Thus the Rl callus line is regarded as partially habituated. Subculture in agitated liquid medium also resulted in the production of large numberr of polyploid cells but these did not persist over the long periods of subsequent growth on agar medium, Enhanced auxin production by the establihed Rl callus line was thus observed in the absence of a detectable level of polyploidy.     

Somatic embryogenesis inGnetum ula Brongn. (Gnetum edule) (Willd) Blume

Summary Somatic embryos ofGnetum ula (Gnetum edule) an endangered gymnosperm closely related to the angiosperms have been induced in vitro. Megagametophyte tissue with immature embryos was cultured on Murashige and Skoog medium. A mucilaginous, translucent embryogenic callus was obtained with 5 mg/l BA. Callus induced with 2,4-D was non-embryogenic. The embryogenic callus in liquid half strength Murashige and Skoog medium without inorganic nitrates supplemented with 2.5 g/l casein hydrolysate and 0.5 g/l L-glutamine gave rise to immature embryos. The embryos matured when treated with 60 g/l sucrose and 10 mg/l abscisic acid.Abbreviations MS Murashige and Skoog - BA 6-benzylaminopurine - 2,4-D 2,4 - dichlorophenoxyacetic acid - ABA abscisic acid     

Mass propagation of <Emphasis Type="Italic">Plectranthus bourneae</Emphasis> Gamble through indirect organogenesis from leaf and internode explants

The present study describes the plant propagation via indirect organogenesis from in vitro derived leaf and internode explants of Plectranthus bourneae, an endemic plant to south India. Leaf and internodal explants successfully callused on Murashige and Skoog medium (MS) supplemented with different concentrations of auxins [2,4-D (2,4-dichlorophenoxyacetic acid), NAA (α-naphthalene acetic acid), IAA (indole-3 acetic acid), IBA (indole-3-butyric acid) and PIC (Picloram); 0.1–2.0 mg/l] in combination with BA (6-benzyladenine) (0.5 mg/l). Maximum callus induction (98 %) was achieved from leaf explant followed by internodal explant (89 %) at 1.0 mg/l NAA, 0.5 mg/l BA. Leaf derived callus showed better shoot regeneration (29.71 shoots) on MS medium containing 1.0 mg/l KN (kinetin), 0.7 mg/l NAA, and 50 mg/l CH (casein hydrolysate) followed by internodal callus (19.71). A maximum of 19.14 roots/shoot was observed at 1.0 mg/l IBA. The rooted plantlets were successfully hardened and transferred to greenhouse condition with 80 % survival. This system could be utilized for large-scale multiplication of P. bourneae by tissue culture.     

Factors affecting shoot initiation from tuber discs of potato (Solanum tuberosum)

Discs of cortical and perimedullary tissue from tubers of potato (Solanum tuberosum L. cv. Superior) formed adventitious shoots when cultured on a medium containing Murashige and Skoog's salts, myo-inositol, 100 mg/l; folic acid, 0.5 mg/l; D-biotin, 0.05 mg/l; gibberellic acid (GA₃), 0.5 mg/l; thiamine ˙ HCl, 0.1 mg/l; glycine, 2.0 mg/l; pyridoxine ˙ HCl, 0.5 mg/l; nicotinic acid, 0.5 mg/l; sucrose, 25 g/l; casein hydrolysate, 1 g/l; Bacto agar, 9.0 g/l; and a cytokinin [N⁶-benzylaminopurine (BAP), N⁶-γ,γ-dimethylallylaminopurine (2iP), or N⁶-furfurylaminopurine (kinetin)]. Discs of pith tissue either failed to survive or produced callus but did not undergo morphogenesis. Cytokinin was essential for explant survival, while BAP at 3.0 mg/l was most efficacious in promoting shoot initiation. Auxin was not essential for shoot initiation or development; however, a low concentration (0.03 mg/l) of α-naphthaleneacetic acid (NAA) stimulated both explant survival and the number of shoots produced per disc. Indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA) did not stimulate shoot initiation. GA₃ was essential for both shoot initiation and subsequent shoot development. The highest incidence of morphogenesis (over 100 shoots in 12 weeks) occurred from tuber discs cultured at 18°C constant and under a photon flux density of 60 μE m^-2s^-1. No difference in regenerative ability was found in explants taken from source tubers stored for 0 to 20 weeks at 4°C. A histological examination indicated that shoots developed from small clusters of meristematic cells which were initiated from within small protuberances on the surface of the tuber disc 3 weeks after transfer.     

Plant regeneration via somatic embryogenesis in mature embryo-derived callus culture of Sinocalamus latiflora (Munro) McClure

Somatic embryogenesis and subsequent formation of plantlets was achieved from callus cultures derived from mature zygotic embryos of Sinocalamus latiflora (Munro) McClure (Bamboo). Embryogenic callus was initiated on Murashige and Skoog's medium (MS) supplemented with 6 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 3 mg/l kinetin, 250 mg/l polyvinylpyrrolidon and 5% sucrose. Prolonged culture of the embryogenic callus on the same medium resulted in embryoid formation. The embryoids developed further to yield whole plantlets when transferred to a medium containing lower concentrations of 2,4-D (3 mg/l) and kinetin (2 mg/l).     

Thede novo Formation of Buds and Plantlets from Various Explants ofAilanthus altissima Mill. Culturedin vitro

The hypocotyls, cotyledons, leaf blades, whole leaves and petioles of seedlings ofAilanthus altissima are capable of producing callus and budsin vitro. Buds and callus were also obtained from whole leaves and internodes of 2-years old plantlets grownin vitro. From the calli buds differentiated and later, both from buds developing directly without a callus phase and alsovia a callus phase, well developed shoots were formed. The cultures were mainained on MS medium in 2 combinations: A) IAA - 0.2 mg 1⁻¹, BAP - 1 mg 1⁻¹, GA₃ - 0.5 mg 1⁻¹, thiamine - 4 mg 1⁻¹ and sucrose 3 %; B) BAP - 0.5 mg 1⁻¹, IAA - 1 mg 1⁻¹, casein hydrolysate 400 mg 1⁻¹, thiamine 4 mg 1⁻¹ and sucrose 3 %. Excised shoots, which had developedde novo in culture, produced roots when incubated on the basic mineral medium of MS with the addition of IAA. The regenerative potential ofA. altissima is very high and this woody species seems to be an ideal object for various morphogenetic studies.     

Induction of somatic embryogenesis in Azadirachta indica

A modified culture protocol has been developed for the induction of somatic embryogenesis in Azadirachta indica (neem). Embryogenic calluses were initiated from cotyledons or hypocotyls using a Murashige and Skoog (MS) agar medium supplemented with 0.5 mg l⁻¹ α-napthaleneacetic acid (NAA), 1 mg l⁻¹ 6-benzylaminopurine (BA), 1 g l⁻¹ casein hydrolysate, and 50 g l⁻¹ sucrose. The calluses, when transferred to a liquid medium similar to the agar medium but with NAA replaced by 0.5 mg l⁻¹ indole-3-acetic acid (IAA), formed globular structures which further developed a rudimentary root, after 4 to 5 weeks incubation. Subsequently, these highly differentiated tissues when transferred into a hormone-free MS medium containing 1 g l⁻¹ casein hydrolysate and 50 g l⁻¹ sucrose, active embryo masses started to appear after 1 to 2 weeks. The embryo production was found to improve more than 2 fold by adding 0.2 mg l⁻¹ zeatin to the medium. This revised version was published online in June 2006 with corrections to the Cover Date.     

ATTEMPTS TO OBTAIN BACTERIA-FREE PLANTS OF PSYCHOTRIA PUNCTATA (RUBIACEAE): GROWTH AND ROOT FORMATION IN CALLUS CULTURES

Attempts were made to obtain bacteria-free plants of Psychotria punctata from tissue cultures. Stem explants and callus derived from them were induced to form roots but failed to form buds on Linsmaier and Skoog medium and 96 chemical modifications of it, including most of those known to induce bud formation in other species. Roots formed with ample IAA (2 mg/liter or more) and a low kinetin concentration (0.25 or 0.50 mg/liter). Adenine inhibited root formation in these media, but tyrosine did not. Tyrosine did lower the percentage of calluses commencing growth. When enzyme-hydrolyzed lactalbumin (1.3 g/liter), kinetin (0.5 mg/liter) and IAA (5 mg/liter) were added to Linsmaier and Skoog medium modified by decreasing inorganic nitrogen and increasing inorganic phosphate, callus grew at the fastest rate observed (increasing threefold in fresh weight in three weeks) and formed numerous roots. This was adopted as the stock callus medium. Casein hydrolysates also stimulated growth but less so than lactalbumin hydrolysate. When lactalbumin hydrolysate or a casein hydrolysate lacking tryptophan was supplied, growth occurred without added auxin if sufficient cytokinin was added. Cytokinin was required at unusually high concentration and was tolerated at still higher concentration. Formation, elongation, and branching of roots persisted on a saturated solution of BA which inhibited callus growth about 70 % and delayed callus senescence. Light caused earlier callus senescence after growth had ceased but did not affect callus growth or root formation. Light-induced senescence was prevented by a high cytokinin concentration.     

Morphogenic potential of mechanically isolated single cells from Digitalis obscura L. callus

Calli from hypocotyl and root explants of Digitalis obscura L. showed regeneration of adventitious shoots, roots and embryos when transferred to Murashige & Skoog medium supplemented with cytokinins alone or in combination with auxins. Optimum shoot-bud formation was achieved in the presence of IAA and BA, while roots mainly appeared either in absence of growth regulators or with IAA and Kn. Embryo formation took place only in those combinations that included Kn. Embryo development was influenced by the type of auxin, and precocious germination occurred in media with NAA. Mechanically isolated cells from hypocotyl- and root-derived calli were plated in MS medium supplemented with several IAA and BA combinations. Single cells were able to proliferate forming callus within 20–30 days in culture. In order to induce organogenesis, calli were transferred to various regeneration media. Shoot-bud differentiation efficiency depended on both callus origin and medium initially used for cell culture, best results being obtained in calli grown from hypocotyl-derived cells cultured in the presence of casein hydrolysate. A further subculture to medium containing coconut milk and lower concentrations of NH₄NO₃ and sucrose promoted shoot development. Rooting was readily achieved upon transferring shoots onto half-strength MS medium. Plantlets were ultimately established in soil.Abbreviations BA benzyladenine - BM basal medium - CH casein hydrolysate - CM coconut milk - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - Kn kinetin - MS Murashige & Skoog - NAA naphthaleneacetic acid     

Plantlet regeneration through indirect shoot organogenesis and somatic embryogenesis in Justicia gendarussa Burm. f., a medicinal plant

A protocol for the regeneration of a large number of plantlets via indirect shoot organogenesis and somatic embryogenesis has been developed from the stem and leaf explants of Justicia gendarussa Burm. f. The callus was efficiently induced from the explants using Murashige and Skoog (MS) medium supplemented with α-Naphthalene acetic acid (NAA) + Benzyl amino purine (BAP) (1.0?+?0.1 mg/l). The highest number of plantlets through indirect shoot organogenesis was obtained when the callus was subcultured to MS medium with BAP + NAA (0.1?+?1.0 mg/l). The maximum number of plantlets via somatic embryos was obtained in the medium with BAP + NAA (1.0?+?0.1 mg/l) for stem derived calli and Kinetin (Kn) + NAA (2.0?+?0.1 mg/l) for leaf derived calli. The in vitro developed shoots were rooted well in half strength MS medium supplemented with 0.5 mg/l of Indole-3-acetic acid (IAA). The in vitro regenerated plantlets were hardened using a mixture of sterile sand:soil:manure (1:1:1). The present study is the first report on the regeneration of plants through somatic embryogenesis from stem and leaf derived calli of J. gendarussa.     

HISTOLOGY OF SOMATIC EMBRYOGENESIS IN PANICUM MAXIMUM (GUINEA GRASS)

Immature embryos of Panicum maximum Jacq. (Guinea grass) were cultured on Murashige and Skoog's medium containing 2,4-dichlorophenoxyacetic acid (5 mg/1) and coconut milk (5%). The epidermal and subepidermal cells of the scutellum at the coleorhizal end either formed somatic embryos directly or gave rise to embryogenic callus at several loci. Somatic embryos were formed later also at the periphery of these embryogenic calli. The embryoids originated from single, densely staining, nonvacuolated and starch-containing cells with thickened walls and developed through characteristic stages of grass embryogeny.     

SOMATIC EMBRYOGENESIS AND PLANT REGENERATION IN TISSUE CULTURES OF PANICUM MAXIMUM JACQ.

Callus tissue cultures were initiated from immature embryos, mature embryos and young inflorescences of Guinea grass (Panicum maximum Jacq.) on Murashige and Skoog's (MS) medium supplemented with 2.5–10 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). Calluses were transferred onto the same nutrient medium with 0.2 mg/l 2,4-D, or without 2,4-D. In callus cultures derived from immature embryos and young inflorescence segments, plantlets were produced via somatic embryogenesis after 3–5 wk. Young plants were successfully transplanted to pots and grown in the greenhouse. Plant development in callus obtained from mature embryos took place through the organization of shoot meristems. Regenerated plants were shown to have the normal tetraploid chromosome number of 2n = 4x = 32.     

Plant regeneration from root callus protoplasts of sour cherry (Prunus cerasus L.)

Summary Callus protoplasts of sour cherry clone CAB4D entered sustained division in Murashige and Skoog's (1962) medium with 1-naphthaleneacetic acid, 6-beneylaminopurine and zeatin. Further to callusing, organogenesis was induced from the protoplastderived callus, in a basal regeneration medium with these same growth regulators at 0.01 mg/l, 2,0 mg/l and 0.05 mg/l, respectively. The regeneration pathway, from such callus, could be altered by adding different organic compounds to this medium. Casein hydrolysate, added alone, promoted rhizogenesis, with shoot buds regenerated from the protoplast-derived roots, while in a basal regeneration medium with casein hydrolysate and a group B vitamin mixture direct caulogenesis occurred.Abbreviations BAP 6-benzylaminopurine - BR basal regeneration medium - CEH casein enzymatic hydrolysate - FPE final plating efficiency - fwt fresh weight - IPE initial plating efficiency - MES 2-N-morpholinoethane sulfonic acid - MPE intermediate plating efficiency - MS Murashige and Skoog (1962) - NAA 1-naphthaleneacetic acid     

Nutritional and Hormonal Requirements for the Growth of Vigna sinensis Callus in vitro

Nutritional and hormonal requirements for in vitro growth of callus tissue of Vigna sinensis Endl. were studied. Callus was formed on hypocotyl and root sections, when they were cultured on Linsmaier and Skoog's basal medium solidified with 10 g/1 agar and supplemented with only 0.5 mg/12,4-D or 2 mg/1 IAA. Further addition of 0.2 mg/1 kinetin and 1 g/l yeast extract resulted in more active callus formation. For unlimited vigorous growth of subcultured callus which was originally isolated from root sections, yeast extract was indispensable besides 2,4-D and kinetin. Such growth-promoting activity was observed also in malt extract and Ebios (dried cell powder of brewery yeast). Of known compounds tested, nicotinic acid, nicotinamide., methyl nicotinate and NAD were promotive to the growth of the callus, although much less effective than yeast extract. Other pyridine derivatives, vitamins and amino acids tested were ineffective or slightly effective. Sucrose was the most suitable carbon source. Fructose, glucose and maltose also supported the growth. Kinetin stimulated cell proliferation of the callus and cell differentiation to tracheary element.