Embryoid and Plantlet Formation from Stock Cultures of Nigella Tissues

Embryogenesis could be induced within a short period in differentiated roots of Nigella sativa on solid Murashige and Skoog's medium containing casein hydrolysate (500 mg/1) and IAA (0.5 mg/1). Experimental root explants were taken from two different stocks: (i) previously differentiated from leaf callus and now being maintained through subcultures in liquid White's medium and (ii) freshly differentiated from leaf callus on solid Murashige and Skoog's medium + casein hydrolysate (100 mg/1) + IAA (0.5 mg/1). Fifty per cent of the embryoids produced 2n plantlets within 40–60 days.     

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 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).     

In vitro Post-germination Growth and Development of Embryos of Alectra (Scrophulariaceae)

In vivo, seeds of the obligate root parasite Alectra vogelii Benth. (Scrophulariaceae) germinate only after being soaked in water for a period of time (pretreatment) followed by stimulation by certain factors exuded from a host root. Germinated seedlings do not develop beyond radicle emergence, and finally die, unless their radicles make contact with and penetrate into a host root conductive system. In vitro, germinated embryos obtained by exposing sterilized and pretreated seeds to root exudate of Vigna unguiculata were aseptically cultured on Knop's, White's and Murashige and Skoog's media. The embryos grew into seedlings with shoots and roots on a medium containing mineral salts and sucrose, but not on mineral salts alone. Seedling performance was generally not improved when the mineral salts-sucrose media were supplemented with vitamins. Shoot extension growth was better on Murashige and Skoog's mineral salts-sucrose medium than on Knop's or White's medium. However, seedling development was greatly boosted when cultivated on White's minerals salts-sucrose medium supplemented with coconut milk. Seedlings turned green on transfer to light but did not flower. The successful culture of these embryos and seedlings on a simple, chemically defined medium of mineral salts and sugar suggests that these nutrient components are the minimal external requirements for stimulation and support of normal seedling growth. These may be obtained in vivo by the parasite's tapping of the host root conductive system.     

Morphogenesis and plant regeneration from cotyledonary cultures of Eucalyptus

Callus cultures were established from hypocotyls and cotyledons derived from young seedlings of Eucalyptus citriodora. Successful plantlet production from cotyledonary callus was achieved within 6 weeks on Murashige and Skoog's basal medium supplemented with zeatin (1 mg/l) and indoleacetic acid (0.2 mg/l). Leaf and shoot callus obtained from one-year-old plants did not differentiate. Results reported contribute to defining optimal conditions for callus growth and plantlet formation.     

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.     

PROLIFERATION AND PLANT REGENERATION FROM THE NODAL REGION OF ZEA MAYS L. (MAIZE,GRAMINEAE) EMBRYOS

Immature embryos of Zea mays L. (maize), cultured on Murashige and Skoog's medium containing 2,4-dichlorophenoxyacetic acid (2,4-D, 0.25–1.0 mg/l) and sucrose (3, 6 and 12%), produced a narrow ridge of tissue in the nodal region of the embryo axis, opposite the scutellum. Only those embryos which were placed with the scutellum in contact with medium showed such a response. The ridge of tissue further proliferated and formed a compact and opaque callus that appeared similar to the scutellar callus. Morphogenesis took place on media containing low concentrations of both 2,4-D and sucrose. Plant regeneration was achieved and is interpreted to take place by the precocious germination of somatic embryos. It is suggested that the ridge of tissue formed at the node may represent an evolutionarily extinct epiblast of the maize embryo and may be cotyledonary in nature.     

ESTABLISHMENT OF LONG-TERM CALLUS CULTURES FROM MATURE WHITE PINE (PINUS STROBUS,PINACEAE)

Callus cultures were established from young vegetative shoots of 15 to 18-year-old white pine trees. The callus has been repeatedly subcultured for 15 months on a modified Murashige and Skoog's medium supplemented with 2 mg/L 6-Benzyladenine and 0.2 mg/L α-Naphthaleneacetic acid. Best callus proliferation occurred when 2 to 4-week-old shoots were used as expiants. Older shoots were heavily contaminated with microorganisms and could be only partially disinfected by heat or chemical treatments.     

In vitro culture of young floral buds of tomato (Lycopersicon esculentum mill.)

Young floral buds of tomato (Lycopersicon esculentum Mill.), with sepal primordia only, were cultured successfully in a liquid medium containing Murashige and Skoog's salts, White's vitamins and glycine, 3% (w/v) sucrose and benzylaminopurine (BAP). In 5 weeks, 60–70% of the cultured buds showed normal flower development through to anthesis, and a full complement of floral organs was produced. In the anthers, microsporogenesis occured and microspores and pollen grains were formed. The gynoecium was differentiated into an ovary, style and a stigma, and the ovary contained many ovules, some of which had developing embryo sacs. The floral organs grown in vitro were, however, smaller in size than those produced in vivo. In the control medium, i.e. without BAP, the floral buds produced petal and stamen primordia only and they too showed limited growth. The data presented shows the importance of cytokinins in the initiation and development of organs in the excised floral buds.     

Tissue Culture of Maize I. Callus Induction and Growth

Callus induction and subculture was successful with mature embryos and stem sections of seedlings of Zea mays L. on Linsmaier and Skoog's medium modified to contain 4 mg/I of 2,4-D and 1 g/I of casamino acids. — 2,4-D was superior to NAA and IAA for both callus induction and growth. Callus subcultured on NAA formed abundant roots on agar-solidified media and numerous root-like primordia in liquid cultures. — Kinetin had no effect on callus induction in the presence of 2,4-D and neither kinetin nor gibberellic acid stimulated callus growth during subculture. — Callus grew equally well on the medium of Linsmaier and Skoog, that of Schenk and Hildebrandt, and the B-5 medium of Gamborg and Eveleigh containing 2% sucrose, 4 mg/I of 2,4-D and 1 g/I of casamino acids. — The callus grew more rapidly at 25°C than at 30°C or 35°C. Little difference was noted at any temperature in callus growth in alternating light (16 h) and dark (8 h) or continuous dark. — Sucrose was superior to glucose and maltose in both liquid and agar-solidified cultures. Lactose and galactose failed to support callus growth.     

Growth and Morphogenesis in Tissue Cultures of Anagallis arvensis

Morphogenesis has been induced in excised organs and callus tissue cultures obtained from various parts of the seedling and mature plants of pimpernel (Anagallis arvensis). Vigorously growing cell cultures capable of being periodically subcultured have been established in liquid as well as on the agar-solidified Murashige and Skoog's medium supplemented with 2,4-D (0.1 mg/1) + kinetin (0.1 mg/l) + coconut milk (10%). The callus tissue obtained from excised hypocotyl segments is white, soft, friable and fast growing, and has been subcultured over a period of two years without showing any sign of decline in growth. The optimum conditions for growth are at pH 5.9, temperature 27°C, and with 4% sucrose as the carbon source. Under appropriate nutritional supply these cultures can be manipulated to induce rhizogenesis in the suspension cultures, and buds and “embryo-like” structures on agar-solidified media. The excised leaves, hypocotyl and stem segments regenerate buds. Of the cytokinins used, 6-(y,y-dimethylallylamino)-purine proved to be the best for the number of cultures producing buds, as well as for the number of buds per culture. Anatomical studies revealed that buds arise from the epidermal and subepidermal layers of leaves and hypocotyl; these buds form shoots which eventually develop into plantlets.     

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.     

MORPHOGENETIC STUDIES ON HAWORTHIA: ESTABLISHMENT OF TISSUE CULTURE AND CONTROL OF DIFFERENTIATION

Investigations were carried out on the in vitro morphogenetic responses of inflorescence segments and gynoecia of several species of Haworthia (Liliaceae). Morphogenetic responses of explants were not species specific. It was found that coconut milk was essential for the growth and differentiation of Haworthia tissue if White's basal medium was used. However, growth and differentiation could be supported by a modified Murashige and Skoog's medium, without any supplements. The investigations demonstrated’ the importance of inositol and ammonium nitrogen in the nutrition of Haworthia tissue cultures. A chemical control of callusing and shoot and root differentiation was obtained by providing appropriate amounts of auxin and cytokinin in the culture medium.     

Efficient plant regeneration from seedling explants of two commercially important temperate eucalypt species–Eucalyptus nitens and E. globulus

A reliable and reproducible method for plant regeneration in vitro of two important temperate eucalypts, Eucalyptus nitens and E. globulus, has been developed which utilises seedling explants. Highly regenerative callus was obtained from individual cotyledon and hypocotyledon explants of both species following cultivation on Murashige and Skoog’s (MS) basal nutrient medium supplemented with 30 g l⁻¹ sucrose, 5–10% (v/v) coconut water, 0.8% agar, 1 mg l⁻¹ -naphthalene-acetic acid (NAA) and 0.5 mg l⁻¹ N⁶ benzylaminopurine (BAP). Shoot differentiation was observed 7–8 weeks after transfer of callus onto regeneration medium containing 0.5 mg l⁻¹ NAA and 1 mg l⁻¹ BAP. In a few instances, direct shoot regeneration occurred without an intervening callus phase in both species. The frequency of plant regeneration was higher for callus derived from hypocotyl segments (30–35%) compared to cotyledonary explants (20–25%) though the average number of shoots per cotyledonary explant was generally higher than for hypocotyl explants. Somatic embryos were observed occasionally in E. nitens, arising from the surface of organogenic callus. Organised structures closely resembling somatic embryos were also observed in E. globulus. Regenerated shoots (30–40%) of both species could be rooted in modified MS media containing indole-3-butyric acid (IBA) and plantlets were successfully transferred to soil.     

Istogenesi e caratteri anatomici dei tessuti ricavati da frammenti di germoglio di Solanum Tuberosum L. coltivati In Vitro

Abstract

HISTOGENESIS AND ANATOMICAL CHARACTERISTICS OF PORTIONS OF SOLANUM TUBEROSUM SPROUTS CULTIVATED IN VITRO. — Disks cut from sprouts of Solanum tuberosum L. have been cultured on White's basic or modified medium (White, 1943) with 20 mg/l KH2₂PO₄ 50 mg/l adenine, and 12 or 24 mg/l NAA.

Sections were prepared according to Morel's method (1948). Only on the disks cultured on White's modified medium with 12 mg/1/NAA growth glomerules and a normal meristematic layer were present, while there were no signs of cellular hypertrophy and organogenetic phenomena.

12 mg/l appears therefore to be the optimal dose of NAA for cultures of excised portions of potato sprouts. Adenine is however always necessary because it triplicates the growth activity of the tissues and because through its antagonism with regard to auxin it favours the genesis of the callus.     

Isolation,culture and plant regeneration of colt cherry (Prunus avium × pseudocerasus) protoplasts

Large numbers of viable protoplasts were isolated from leaf mesophyll tissues and cell suspension cultures of Colt cherry using 1% (w/v) Onozuka R-10, 0.2% (w/v) Macerozyme R-10, 0.1% (w/v) Driselase, 1% (w/v) polyvinylpirrolidone (av. MW 10 000) (PVP-10) and 2% (w/v) Meicelase, 2% (w/v) Rhozyme HP-150 and 0.03% (w/v) Macerozyme R-10. Culture media, based on Murashige and Skoog's (MS) salts, supplemented with 9% (w/v) mannitol and various combinations of α-naphthalene acetic acid (NAA), 6-benzylaminopurine (BAP) and zeatin (Z) promoted cell wall regeneration followed by cell colony and callus formation. Protoplasts of both sources were compared in relation to their cultural requirements. Protoplast-derived callus underwent organogenesis.     

Somatic embryogenesis and subsequent plant regeneration from inflorescence callus of Bambusa beecheyana Munro var. beecheyana

Somatic embryos of bamboo, Bambusa beecheyana Munro var. beecheyana were developed in callus derived from young florets and adventive roots obtained from floret callus. The medium was a modified Murashige and Skoog medium (1962) supplemented with 3 mg/l 2,4-dichlorophenoxyacetic acid, 2 mg/l kinetin, a high content of sucrose (6%) and 0.7% agar. The embryoids germinated spontaneously to yield whole plantlets on this medium with or without the hormonal adjuvants.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - NAA naphthaleneacetic acid - MS Murashige and Skoog's (1962) medium     

Somatic embryogenesis in two Gossypium hirsutum genotypes on semi-solid versus liquid proliferation media

A comparison of semi-solid vs. liquid embryo proliferation media was made using two Gossypium hirsutum L. genotypes (Coker 312 and T25) and two callus initiation media. Sections of petioles from mature, flowering plants were cultured on two modified Murashige and Skoog media. Medium 1 included 4.0 mg l^-1 NAA and 1.0 mg l^-1 kinetin; medium 2 contained 0.1 mg l^-1 2,4-D and 0.1 mg l^-1 kinetin. After six weeks, callus was removed from each explant and divided in half. One callus portion was placed in liquid proliferation medium and the other on semi-solid (0.2% Gelrite) proliferation medium. Composition of proliferation medium was identical to that of initiation medium, except no growth regulators were added. Embryos were counted after eight weeks. The percentage of explants forming callus was influenced by genotype/initiation medium combination. Analysis of variance procedures revealed significant variability for callus initiation media, proliferation media (semi-solid or liquid), and an initiation medium x genotype interaction. Paired t-tests indicated that more embryos were produced in liquid proliferation medium (227.3 embryos/culture) than on semi-solid proliferation medium (134.6 embryos/culture).Abbreviations NAA naphtaleneacetic acid - 2,4-D 2,4-D dichlorophenoxyacetic acid     

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.     

Improved plant regeneration in callus cultures of Sorghum bicolor (L.) Moench

Sorghum bicolor is a recalcitrant species for tissue culture regeneration and genetic transformation. Browning of explants is one of the factors limiting organ and tissue cultures. To overcome this, callus tissue was initiated from the shoot tips of in vitro germinating seeds (S. bicolor cv. Róna 1), and then cultured on modified MS media (Murashige and Skoog in Physiol Plant 15:473–497, 1962). In the first experiment, we tested callus induction on several media supplemented with casein hydrolysate, polyvinylpyrrolidone, honey, and sucrose. The best callus induction was recorded for the medium with honey and sucrose (80.0%) and for control medium (79.8%). Shoot regeneration was tested on the MS medium with 6-benzylaminopurine (BAP) supplemented with honey and sucrose at a 1:1 ratio (by weight) or with sucrose only. The highest percentage of calluses regenerating shoots was noted for those induced on the medium with sucrose and honey—approx. four times higher when compared to the control. Rooted plantlets were acclimatized with a 92% survival rate. In the second experiment, we analyzed culture responses to various ways of honey application to the induction media: honey (autoclaved or filtered) in presence or absence of sucrose. Supplementation of the medium with fructose, glucose, and maltose at a proportion typical for honey was also investigated. The explant and callus survival rates were similar to those of the honey–sucrose combination in the first experiment. Only presence of both sucrose and honey in the induction medium improved the total regeneration rate to 37.9% over the control (18.8%). Sucrose and honey appear to act synergistically for shoot regeneration in callus cultures of sorghum.