In vitro callus induction and regeneration studies in Withania somnifera

Callus cultures were initiated from axillary leaves, axillary shoots, hypocotyls, and root segments on Murashige and Skoog (MS) (1962) medium supplemented with 2,4-D (2 mg l⁻¹) and KN (0.2 mg l⁻¹). Shoots differentiated best from axillary shoot base callus on MS medium containing BA (2 mg l⁻¹). Regenerated shoots rooted best on MS medium containing IBA (2 mg l⁻¹) alone, and IBA (2 mg l⁻¹) with IAA (2 mg l⁻¹). Plantlets were transferred to pots containing sand and soil mixture, acclimatized in a culture room and afterwards transferred to the glasshouse. This revised version was published online in June 2006 with corrections to the Cover Date.     

Shoot apical meristem: In vitro regeneration and morphogenesis in wheat (Triticum aestivum L.)

Summary We report a less genotype-dependent in vitro regeneration system capable of producing multiple shoot clumps and whole plants in four different wheat genotypes. Shool apical meristems from 7-d-old-seedlings produced axillary and adventitious shoots and somatic embryos on media containing N⁶-benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid (2,4-D). All four genotypes responded positively to shoot multiplication depending upon media composition. Scanning electron microscopies of cultures showed a proliferating budding state that gave rise to adventitious shoots and somatic embryos on further multiplication. The percentage of relative shoot apical meristem multiplication was 80–90%, and the average number of shoot meristems per multiplied shoot was 40–50 in all genotypes. Among different concentrations of phytohormones, 2 and 4 mgl⁻¹ BA (8.8 and 17.7 μM) in combination with 0.5 mg l⁻¹ 2,4-D (2.26 μM) gave the best results. Actively multiplying shoot clumps were recovered with high frequency among 3-mo.-old cultures. These shoot clumps regenerated normally and produced fertile plants containing viable seeds. This in vitro system might prove useful for the production of transgenic plants of wheat in a relatively genotype-independent manner.     

In vitro regeneration in Allium species

An attempt to induce shoot regeneration from leaf disc explants from Allium sativum L., A. porrum L., and A. schoenoprasum L. and the induction of shoot regeneration from single flower-bud receptacles in A. porrum is presented. While the regeneration rate from leaf disc explants was low, an efficient method for propagating A. porrum in vitro was obtained by cultivating single flower-bud receptacles. The shoot regeneration ability was strongly controlled by the genotype. Up to 294 shoots per leek plant could be harvested. Simultaneously the same plant could be used for seed production and bulbil formation in vivo. The efficiency of the in vitro multiplication method described allows the integration of this procedure into breeding programmes of A. porrum Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxy acetic acid - IAA 3-indole acetic acid - NAA 2-naphtalene acetic acid     

In vitro studies on the anatomy and morphology of bud regeneration in melon cotyledons

Summary The anatomy and morphology of bud regeneration were investigated in melon (Cucumis melo L.) cv. Galia, which regenerates in vitro only by direct organogenesis from the cotyledon explant. Explants were cut from the cotyledon proximal to the apex from 3-d-old in vitro seedlings. After 3 d on Murashige and Skoog medium with N⁶-benzyladenine, cell division can be observed in the epidermal layer on the adaxial side in the center of the explant, near the most proximal (wounded) cut edge. Over the next week, the area of the meristem increases laterally. Additional cell layers are added to the meristematic area by cell division in the epidermis. In places the epidermis remains active in cell division. Alongside those active areas there are zones where the epidermis has become inactive, although the subepidermal layers continue to divide. In transverse section, the explant now has small protuberances on the adaxial surface. After 10 d on cytokinin-containing medium, the first signs of development are visible on the adaxial surface adjacent to the proximal cut edge. The protuberances observed after 10 d are neither primordia nor buds, although some meristematic bulges are observed. The first regenerated shoot buds are observed histologically after 15 d, by which time the surface has many protuberances and some small leaves. The first shoot is found by histology after 22 d. By this time the surface is covered with protrusions and leaves, mostly without accompanying buds. The leaves may be produced from the protrusions initially visible after 10 d.     

In vitro morphogenesis ofCrambe maritima L.

Summary Tissue cultures isolated from the root ofCrambe maritima have now undergone 17 transfers over two years on a medium containing 10% coconut milk, 2.0 mg/l IAA + 0.8 mg/l kinetin. The cultures consist predominantly of organized structures varying in complexity from nodular to root-like outgrowths and teratomatous leafy shoots. Embryoid-like structures also occur.     

In vitro morphogenesis of amphibian erythroblasts.

Non-mammalian vertebrate erythrocytes are flattened nucleated ellipsoids containing marginal bands (MBs) of microtubules that assemble during cellular morphogenesis. Earlier work suggested that pointed erythroid cells containing pointed MBs were intermediate stages in terminal differentiation, rather than aberrant forms, but direct evidence was lacking. Here we report on morphogenesis in individual post-cytokinetic amphibian erythroblasts in culture. Daughter cells remained adjacent in pairs, and developed pointed morphology over 1-2 h in the following sequence: (a) ends opposite the cytokinetic furrow became pointed, producing a spheroidal singly-pointed stage; (b) furrow ends usually became pointed, yielding doubly-pointed cells; (c) furrow-end points disappeared, producing a second singly-pointed stage that was flattening. Over a longer term, the single points sometimes disappeared, yielding a flattened discoid. These observations support the hypothesis that pointed cells are normal intermediates in a biogenetic program in which post-mitotic centrosomes organize MBs while occupying the singly-pointed ends of differentiating erythroblasts.     

High frequency plant regeneration from the cotyledonary node of common bean

An efficient regeneration system for Phaseolus vulgaris was developed from mature seeds germinated on Murashige and Skoog (MS) medium supplemented with thidiazuron or N⁶-benzylaminopurine (BA) for 6 d. Using cotyledonary nodes, multiple buds were induced on the MS medium supplemented with 5.0 mg dm⁻³ BA with the induction frequency 71.9 % after 4-week culture. The buds were then transferred onto shoot formation medium containing 1.0 mg dm⁻³ BA, 0.1 mg dm⁻³ gibberellic acid and 2.0 mg dm⁻³ silver nitrate. The addition of AgNO₃ enhanced the frequency of the shoot formation from 61.3 to 87.6 %. Root induction medium was half-strength MS medium with 0.75 mg dm⁻³ indolebutyric acid and 0.02 mg dm⁻³ BA. The average root frequency was 84.3 %. The regenerated plantlets with healthy roots grew successfully when transferred to soil. Using this system we obtained over 10 regenerated plantlets from one explant.     

In vitro regeneration of Trifolium glomeratum

In vitro regeneration of Trifolium glomeratum, a leguminous forage species, was attempted through leaf, petiole, cotyledon, hypocotyl, collar and root explants and two media combinations. Root and collar explants showed no callus induction. Medium with 0.05 mg dm⁻³ α-naphthaleneacetic acid (NAA) and 0.10 mg dm⁻³ N⁶-benzyladenine (BA) was more effective for hypocotyl explant whereas cotyledon and petiole explant were more responsive to 5.0 mg dm⁻³ NAA and 1.0 mg dm⁻³ BA. Friable, green calli obtained from petiole explant on this medium showed organogenetic potential. Modified root-inducing medium having 0.21 mg dm⁻³ indole-3-acetic acid and 2.5 % sucrose was successful for root induction and plantlets were successfully transferred to field after hardening and Rhizobium inoculation.     

In vitro regeneration of Eucalyptus camaldulensis

An efficient in vitro regeneration protocol enables mass multiplication, genetic modification and germplasm conservation of desired plants. In vitro plant regeneration was achieved from nodal segments of 18-months-old superior genotypes of Eucalyptus camaldulensis trees through direct organogenesis (DO) and direct somatic embryogenesis (DSE) pathways. Initial bud break (BB) stage occurred via DO while shoot multiplication phase followed both DO and DSE pathways. Interestingly, both BB and shoot multiplication stages were achieved on shoot induction and multiplication (SIM) media composed of Murashige and Skoog (MS) basal medium supplemented with 2 mg l⁻¹ benzyl aminopurine (BAP) and 0.1 mg l⁻¹ naphthalene acetic acid (NAA). Best shoot elongation response was observed on half strength MS fortified with 0.5 mg l⁻¹ BAP, while root induction and elongation was superior in 1/2 MS + 1 mg l⁻¹ Indole butyric acid (IBA). Full strength MS fortified with cytokinins (BAP) and weak auxin (NAA) in the ratio of 20:1 favored direct regeneration pathways. Further, half strength MS supported shoot and root development. The absence of intervening callus phase in this protocol can help in minimizing the chance occurrence of somaclones. When compared to other compositions tried, hardening in 100 % coco peat resulted in maximum survival (80 %) of the in vitro raised plantlets. For mass multiplication, fortnight subculturing of a single nodal explants for eight passages on SIM medium resulted in 60–148 shoot initials. Repeated subculturing in SIM medium induced the formation of direct somatic embryos which in turn improved the turnover capacity and enabled large scale clonal multiplication of elite and desirable trees of E. camaldulensis. Following this protocol, it takes a minimum time period of four-months between in vitro explant inoculation to hardening stage. In the present study, DO and DSE pathway of plant regeneration was reported occurring simultaneously in the same nodal explants of E. camaldulensis.     

In vitro morphogenesis of chick embryo hypertrophic cartilage

Dedifferentiated chick embryo chondrocytes (Castagnola, P., G. Moro, F. Descalzi-Cancedda, and R. Cancedda, 1986, J. Cell Biol., 102:2310-2317), when transferred to suspension culture on agarose-coated dishes in the presence of ascorbic acid, aggregate and remain clustered. With time in culture, clusters grow in size and adhere to each other, forming structures that may be several millimeters in dimension. These structures after 7 d of culture have the histologic appearance of mature hypertrophic cartilage partially surrounded by a layer of elongated cells resembling the perichondrium. Cells inside the aggregates have ultrastructural features of stage I (proliferating) or stage II (hypertrophic) chondrocytes depending on their location. Occurrence and distribution of type I, II, and X collagens in the in vitro-formed cartilage at different times of culture, show a temporal and spatial distribution of these antigens reminiscent of the maturation events occurring in the cartilage in vivo. A comparable histologic appearance is shown also by cell aggregates obtained starting with a population of cells derived from a single, cloned, dedifferentiated chondrocyte.     

In vitro morphogenesis of foot-and-mouth disease virus.

Foot-and-mouth disease virion RNA is translated efficiently and completely in a rabbit reticulocyte lysate cell-free system. Treatment of cell-free lysates with monospecific serum prepared against the individual viral structural proteins or with monoclonal antibodies prepared against the inactivated virus or against a viral structural protein precipitated all of the structural proteins, suggesting that structural protein complexes were formed in vitro. Sucrose gradient analysis of the cell-free lysate indicated that complexes sedimenting at 5, 14, 60 to 70, and ca. 110S were assembled in vitro. Structural proteins VP0, VP1, and VP3 were the major polypeptides found in these complexes. The material sedimenting at 110S, i.e., containing VP0, VP1, and VP3, was precipitated by a 140S-specific monoclonal antibody but not by a 12S subunit-specific monoclonal antibody, suggesting that this capsid structure contained at least one epitope present on the intact virus.     

In vitro morphogenesis in Selaginella microphylla (Kunth.) Spring

Selaginella, an extant genus of primitive vascular plants, has survived over 400 million years of evolution. In vitro morphogenesis in Selaginella microphylla is considered for the first time to establish a well-documented aseptic culture on half- strength Murashige and Skoog’s basal medium with 2ip (4.92–49.21 μM), or Kn (4.65–46.47 μM) or GA₃ (2.89–28.90 μM) for shoot multiplication, and with different concentrations of IBA (4.9–49 μm) to initiate root cultures. GA₃ was instrumental for shoot multiplication as well as induction of reproductive structures in each and every leaf axil. On the other hand, it is observed that IBA alone in S. microphylla can act as signal molecules for induction of enormous numbers of root masses from a few existing roots. An interesting pattern of re-differentiation has also been observed where apical portions of large numbers of roots were converted to green shoot apical meristems. Further differentiation produced tiny green shoots. Distinct bipolarity was noted in shoots when they were isolated from root masses and appeared as embryo-like structures. Chromosome analysis from in vitro sporophytic plants revealed 2n = 16 chromosomes, indicating chromosomal stability. The interesting in vitro pattern of morphogenesis obtained in S. microphylla may provide new insights into totipotency of plants.     

In vitro regeneration through organogenesis in Egyptian chickpea

Abstract

Genetic engineering for improvement of the recalcitrant crop chickpea (Cicer arietinum L.) was largely restricted by the lack of an efficient regeneration system. In vitro regeneration in two Egyptian chickpea varieties, Giza 531 and Giza 4 was achieved by direct organogenesis. A variety of embryo explants and different types and concentrations of growth regulators were investigated for maximum efficiency of shoot and root regeneration. Embryo axes with the adjacent part of cotyledon proved to be the most promising type of explant for shooting and rooting responses. 6-Benzylaminopurine (BAP) and indole-3-butyric acid (IBA) were found to induce the highest percentages of shoot initiation and root formation, respectively. Although the Giza 531 variety produced a better response than the Giza 4 for shoot formation, it displayed lower performance for root induction. It would be rewarding if this optimized regeneration protocol paved the way toward the genetic improvement of the Egyptian chickpea.     

In vitro regeneration and genetic manipulation of grasses

Regeneration of plants from cultured cells is an important and essential component of plant biotechnology. Advances in the recovery of plants from cultured cells and protoplasts of grasses, and in genetic transformation provide challenging opportunities for the genetic manipulation and improvement of this most important group of food plants.     

In vitro regeneration of plantlets in Brassica alboglabra

Different vegetative parts of Brassica alboglabra seedlings and mature plants were used as explants in culture.A high frequency (60–100%) of shoot regeneration was obtained from hypocotyl explants, nodal stem segments, internodal segments and shoot apices cultured on Murashige-Skoog basal medium. Addition of 6-benzylaminopurine and kinetin increased the average number of shoots per explant. When detached and transferred to basal medium, the shoots readily developed roots. Regenerated plantlets could be successfully transplanted in soil.     

In vitro plant regeneration in the genus Cucumis

Plants were regenerated from cotyledonary and root explants of cucumber (Cucumis sativus L.) cultivars and breeding lines of diverse sex type, growth habit, and processing quality and from cotyledonary explants of muskmelon (C. melo L.). Somatic embryogenesis was induced on a medium consisting of Murashige and Skoog (MS) salts supplemented with 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 1.0 mg/l α-naphthaleneacetic acid and 0.5 mg/l 6-benzylaminopurine. Embryos matured on the same medium without 2,4-D, and developed into normal plants on a hormone-free MS medium. Cucumber plants were also regenerated from cotyledonary protoplasts using a modified tomato protocol.     

In vitro regeneration of apomictic bluestem grasses

Explants from immature inflorescences of four genotypes of Old World bluestem grasses, (Bothriochloa spp.), produced callus tissue on Linsmaier and Skoog (RM) and 1/2 Murashige and Skoog (1/2 MS) media containing high levels of growth regulators. Callus masses were composed of two distinct tissue types, one a compact, white, embryogenic portion (E calli), the other soft, translucent, gelatinous and nonembryogenic (NE calli). When transferred to medium with a reduced level of 2,4-D, and/or supplemented with zeatin, E callus underwent further organization culminating in shoot production. Light and scanning electron microscopy confirmed the embryogenic pathway of differentiation. Genotype significantly affected callus induction frequency and the number of plants regenerated. The RM medium induced more explants to initiate callus compared to the 1/2 MS medium. Age of the inflorescence explant, as indicated by size, was critical for callus induction. Inflorescences with racemes 8 mm in length were superior to older ones. Five-hundred-twenty-two plantlets were regenerated and grown to maturity.     

In vitro morphogenesis of Cucumis melo var. inodorus

In vitro morphogenesis of C. melo L. var. inodorus was studied by the induction of adventitious buds and somatic embryos. Organogenesis was obtained from cotyledon segments and leaf discs in culture medium supplemented with benzylaminopurine (1 mg l⁻¹) and somatic embryogenesis was induced in medium containing 2,4-dichlorophenoxyacetic acid (5 mg l⁻¹) + thidiazuron (1 mg l⁻¹). Through histological analysis it was possible to verify that in cotyledonary explants, protuberances that do not develop into well-formed shoot buds and leaf primordia are more frequently formed than complete shoot buds, resulting in a low frequency of plant recovery in the organogenic process. A high percentage of explants responded with the formation of somatic embryos; the microscopical analysis showed that the somatic embryos lacking well developed apical meristems had a low conversion rate into plants. Plant recovery was not obtained from leaf-disc explants, with high rates of contamination and formation of protuberances which did not develop into shoot buds. Histological sections showed the development of epidermis and leaf hairs, indicating those structures could be leaf primordia; however, these were not associated with a shoot apical meristem. This revised version was published online in June 2006 with corrections to the Cover Date.