In vitro selection and regeneration of cotton resistant to high temperature stress

Summary Cell suspension cultures of cotton (Gossypium hitirsutum L. cv. Coker 312) were exposed to various temperature:time treatments in order to select cell lines resistant to high temperature stress. Cells were exposed to 45°C for 3 h each day until the total accumulated hours of stress were: 0 h, 10 h, 75 h, 100 h, or 105 h (81 h pulsed then 24 h continuous). After the stress treatments, the cells were plated onto embryo development medium and plants were recovered. The embryogenic calli that were recovered were subcultured monthly for 6 months and tested for increased resistance to the temperature:time treatments previously determined to be lethal and to water stress as imposed by PEG. All of the selected cell lines were more resistant to both types of stress than the control cell lines. Leaf tissue of stress selected (Ro) formed and maintained callus growth when incubated at 38°C; whereas, tissue excised from nonselected controls rarely formed callus and calli which did form quickly became necrotic. These cells and plants will provide a tool for determining the mechanisms involved in resistance to high temperature stress.     

In vitro evidence that interactions betweenXenopus blastomeres restrict cell migration

Summary The consistency of the frog blastula's fate map is produced, in part, because the progeny of blastomeres located in dfferent regions do not intermix with one another. We examined the cause for this restriction of intermixing in two types of cultures. In one type of culture, two groups of cells were excised from blastulae and stuck together; the movement of cells between the groups was monitored. Cells migrated more extensively between groups derived from the same region than between groups derived from different regions. In the other type of culture, a single cell was implanted into a group of cells that was excised from the blastula. The rate of division and the extent of migration of the implanted cell's clone were monitored. The implanted cell divided more rapidly among cells from its own region than among cells from a different region. Both experiments show that the restriction of intermixing that occurs between regions of the intact embryo also occurs in vitro. These results indicate that the restriction does not result secondarily from normal morphogenetic movements, which are absent from the explants, but probably from cellular interactions that limit the extent of cell migration. This limitation is correlated with a reduction in the rate of cell division.     

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 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 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 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 Hypoxis colchicifolia plantlets

Corms of Hypoxis species are heavily traded for use in traditional medicine in southern Africa. High demand has increased unsustainable harvesting from the wild, diminishing natural populations. Micropropagation of Hypoxis colchicifolia was investigated as a means of mass producing plants for both commercialization and re-establishment in the wild. Various plant organs were tested as explant sources and decontamination optimized for each explant type. Seeds failed to germinate in vitro, and inflorescence peduncles, leaves (young and mature) turned brown and did not respond in culture. Only 6% of corm explants produced callus and shoots. Flower buds responded best, with multiple shoots initiated from explants either directly from meristemoids or indirectly via callus. The problem of browning due to phenolic exudation was solved by including polyvinylpyrrolidone (PVP) in the culture medium when required. Common pathogens were partially controlled by washing affected explants in benomyl solutions. Rooting and corm induction were successful, and plantlets could be stored at low temperature (10 °C) prior to acclimatization with no adverse effects. In planting trials with 5-month-old and 21-month-old plants regenerated using the improved protocols, flowering percentage, corm and leaf size were increased significantly in plants grown in pots compared to those grown in the field over a 28 month period.     

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 regeneration and morphogenesis studies in common bean

An efficient protocol for high frequency in vitro regeneration of multiple shoots and somatic embryos from the embryonic axis of common bean (Phaseolus vulgaris) was developed. Ten common bean cultivars representing a wide range of diversity among current commercial market classes were used for in vitro regeneration evaluation in our study. These cultivars were tested on 63 different media formulations consisting of combinations of cytokinins, namely benzyladenine (BA) and thidiazuron (TDZ) at concentration levels of 0.0, 1.0, 2.5, 5.0 and 10.0 mg l⁻¹ and auxin, namely naphthalene acetic acid (NAA) and indole-3-acetic acid (IAA) at concentration levels of 0.0, 0.05, 0.1 and 1.0 mg l⁻¹. P. vulgaris cv. Olathe pinto bean performed the best producing over 20 multiple shoots per explant while cv. Condor black bean was the poorest with nine multiple shoots per explant. The optimum media for regeneration of multiple shoots was 4.4 mg l⁻¹ Murashige and Skoog (MS) containing 2.5 mg l⁻¹ BA and 0.1 mg l⁻¹ IAA supplemented with 30 mg l⁻¹ silver nitrate. Adventitious shoots and somatic embryos were regenerated on 4.4 mg l⁻¹ MS medium containing 1 mg l⁻¹ TDZ and 0.05 mg l⁻¹ NAA supplemented with 30 mg l⁻¹ silver nitrate or activated charcoal. Efficient and effective rooting of plantlets was achieved by dipping the cut end base of in vitro regenerated shoots in 1.0 mg l⁻¹ indole-3-butyric acid (IBA) solution and culturing on media containing 4.4 mg l⁻¹ MS supplemented by 0.1 mg l⁻¹ IAA, NAA or IBA.     

In vitro somatic embryogenesis and plant regeneration of cassava

An efficient and reproducible plant regeneration system, initiated in somatic tissues, has been devised for cassava (Manihot esculenta Crantz). Somatic embryogenesis has been induced from shoot tips and immature leaves of in vitro shoot cultures of 15 cassava genotypes. Somatic embryos developed directly on the explants when cultured on a medium containing 4–16 mg/l 2,4-D. Differences were observed with respect to the embryogenic capacity of the explants of different varieties. Secondary embryogenesis has been induced by subculture on solid or liquid induction medium. Long term cultures were established and maintained for up to 18 months by repeated subculture of the proliferating somatic embryos. Plantlets developed from primary and secondary embryos in the presence of 0.1 mg/l BAP, 1mg/l GA₃, and 0.01 mg/l 2,4-D. Regenerated plants were transferred to the field, and were grown to maturity.     

In vitro proliferation of shoots and regeneration of cotton

Shoot proliferation from different explants of several Indian cultivars of cotton was studied in culture. Cotyledonary nodes taken along with the shoot apex of seedlings produced multiple shoots on modified MS nutrient agar supplemented with cytokinins. 6-Benzyladenine was most effective in inducing growth of multiple shoots. Explants of several genotypes formed organogenic masses that differentiated to secondary shoots on repeated subculture. The isolated shoots were rooted on basal medium supplemented with naphthaleneacetic acid and were transferred to soil after acclimatization. This revised version was published online in June 2006 with corrections to the Cover Date.     

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 of evergreen azalea from leaves

Rhododendron simsii Hellmut Vogel was regenerated using different types of explants, auxins and cytokinins. After a callus induction phase, with 2,4-dichlorophenoxyacetic acid or -naphthaleneacetic acid, adventitious shoot regeneration was obtained on a medium supplemented with thidiazuron or zeatin. With thidiazuron shoots were small and a subsequent elongation step was required before rooting. An elongation step was not required when zeatin was used. The duration of the callus induction phase was negatively correlated with the regeneration capacity.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - 2iP 6-(--dimethylallylamino)purine - NOA ß-naphthoxyacetic acid - BA 6-benzyladenine - IBA 1-H-indole-3-butyric acid - IAA 1-H-indole-3-acetic acid - TDZ thidiazuron - WPM woody plant medium     

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 Acacia mangium via organogenesis

Plant regeneration of Acacia mangium was achieved through organogenesis in callus cultures. Calli were induced from five types of explants (embryo axes and cotyledons of mature zygotic embryos as well as leaflets, petioles and stems of seedlings) of A. mangium on MS (Murashige and Skoog, 1962) basal medium containing 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 13.95 μM kinetin (KT). Green or green purple compact nodules containing clusters of meristematic centers were induced in these calli after transfer to MS basal medium containing 1.14–22.75 μM thidiazuron (TDZ) and 1.43–2.86 μM indole-3-acetic acid (IAA). A combination of 4.55 μM TDZ and 1.43 μM IAA promoted the highest percentage of calli to form nodules, in 8–11% of calli derived from cotyledons, embryo axes, leaflets or petiole and in 4% of calli derived from stems. Twenty-two percent of the nodules formed adventitious shoots on MS basal medium containing 0.045 μM TDZ. Shoots were elongated on MS medium containing 0.045 μM TDZ supplemented with 7.22 μM gibberellic acid. The medium containing 10.75 μM NAA and 2.33 μM KT promoted rooting of 10% of the elongated shoots. Plantlets grew up well in the green house. This revised version was published online in June 2006 with corrections to the Cover Date.