B.V. Conger

In vitro differentiation of multiple shoot clumps from intact seedlings of switchgrass

SummaryAn efficient and reproduciblein vitro culture system has been developed for regeneration of multiple shoot clumps from intact seedlings of both lowland and upland cultivars of switchgrass (Panicum virgatum L.). The multiple shoots were induced on Murashige and Skoog medium supplemented with various combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 1-phenyl-3-(1,2,3-thiadiazol-5YL)-urea (thidiazuron or TDZ). Maximum response was obtained with 4.5 μM 2,4-D and 18.2 μM TDZ. These shoots proliferated and rooted efficiently on MS medium without growth regulators. The developmental pattern of the multiple shoots indicated their origin from the enlarged shoot apex via proliferation of axillary buds and subsequent reprogramming of shoot meristems followed by secondary differentiation of adventitious shoots The simplicity of the protocol and direct production of multiple shoots make this a potential system that is highly attractive and amenable for microprojectile-mediated gene transfer.

Influence of different auxins at varying concentrations on callus inductions and growth from embryo and leaf-tip explants in gramineae

Abstract Experiments were conducted to determine the effectiveness of 2,4-dichlorophenxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), α-2,4,5-trichlorophenoxypropionic acid (silvex), p -chlorophenoxyacetic acid ( p CPA), 3,6-dichloro- o -anisic acid (dicamba), 4-amino-3,5,6-trichloropicolinic acid (picloram), α-naphthaleneacetic acid (NAA) and indole-3-acetic acid (IAA) at molar concentrations of 5–60 μM on callus induction and growth of orchardgrass ( Dactylis glomerata L.), tall fescue ( Festuca arundinacea Schreb.) and annual ryegrass ( Lolium multiflorum Lam.) embryo explants. The effectiveness of 2,4-D, 2,4,5-T, silvex, dicamba, picloram and NAA on callus induction from leaf-tip explants was studied in annual ryegrass. Length of leaf from which tips were excised and whether or not the mesocotyl-tip node was included in the explant on the efficiency of callus induction were also studied in annual ryegrass. The three species responded differently with regard to different auxins and auxin concentrations for optimum callus induction and growth from embryos. In general, orchardgrass calli grew faster and attained a larger size during the 28-day incubation period than the other two species. Tall fescue calli exhibited the least growth. Dicamba and picloram were effective for inducing callus in all three species and were especially superior to 2,4-D in tall fescue. These auxins were also effective for inducing callus from leaf-tips in annual ryegrass but higher concentrations were needed than for embryos. Production of callus from leaf-tip was highly variable but tips from shorter leaves gave higher percentage of callus induction than those from longer leaves. Percent callus induction was 88% or above if the node were included in the explant regardless of initial leaf length or dicamba concentration.

Growth, proline accumulation and water relations of NaCl-selected and non-selected callus lines of Dactylis glomerata L.

Sodium chloride-tolerant calli were selected from leaf-derived embryogenic calli of Dactylis glomerata L. on agar solidified medium supplemented with 200 mM NaCl, a concentration lethal to non-selected calli. Growth characteristics, water relations and proline accumulation pattern were compared in selected and non-selected lines. The objective was to gain an understanding of the mechanism(s) of tolerance in the NaCl-tolerant line. Growth in the selected line, as expressed in terms of tolerance index (ratio of fresh wt. on NaCl medium:fresh wt. on NaCl free medium x 100), was greater than that of the non-selected line at all levels of NaCl between 50 and 300 mM. There was no significant difference in proline accumulation in the selected and non-selected lines. Maintenance of turgor by osmotic adjustment was observed in the non-selected line despite decreased growth. In contrast, the selected line lost either the need or the ability to adjust osmotically. There was little or no increase in symplastic osmolality in the selected line when exposed to NaCl. Presumably, selection was made for a salt-excluding tissue that has lost the ability to accumulate solutes and adjust turgor with NaCl stress.

Relationship of abscisic acid to somatic embryogenesis in Dactylis glomerata

Abstract The influence of abscisic acid (ABA) on somatic embryogenesis in orchardgrass (Dactylis glomerata L.) was studied in one embryogenic (Embryogen-P) and one nonembryogenic (I-39) genotype. Leaf segments from the basal portions of the two innermost leaves were cultured for various lengths of time on SH medium containing 0.01–20.0 μM ABA. Somatic embryogenesis was increased in Embryogen-P by a 3-day exposure to 0.01–10.0 μM ABA; 20 μM was inhibitory. I-39 did not respond to any ABA treatment. Quantification by enzyme-linked immunosorbent assay (ELISA) indicated that exogenous ABA levels in leaf tissues were nearly the same in the two genotypes at the initiation of culture but decreased much more rapidly during the first 7 days in the nonembryogenic than in the embryogenic genotype. They were the same after 14 days of culture. These results suggest that somatic embryogenesis in the embryogenic genotype may be increased by manipulation of concentration and duration of ABA application. Attempts to induce somatic embryogenesis in the nonembryogenic genotype with ABA treatments were not successful.

Comparison of 2,4-D and 2,4,5-T on callus induction and growth in three Gramineae species

Abstract The synthetic auxins 2,4-D and 2,4,5-T were compared at concentrations ranging from 10 μM to 60 μM on callus induction and growth of orchardgrass (Dactylis glomerata L.), annual ryegrass (Lolium multiflorum Lam.) and tall fescue (Festuca arundinacea Schreb.). This comparison was made on whole caryopses of orchardgrass and on embryos of all three species. Culture was on a modified Murashige-Skoog (MS) medium at 26°C in the dark for 19 days (whole caryopses) or 42 days (embryos). Both auxins were effective in inducing callus in both whole caryopses of orchardgrass and embryos of all three species. Higher auxin concentrations, especially of 2,4-D, were required to induce maximum callus growth for whole caryopses than for embryos of orchardgrass. Except for fescue embryos, good callus growth was induced by 2,4,5-T and lower concentrations of this auxin than 2,4-D were required for maximum growth. The effective concentration range of 2,4,5-T, however, appeared to be narrower than that of 2,4-D. Annual ryegrass embryos tolerated higher concentrations of 2,4,5-T before growth inhibition than orchardgrass embryos. The amount of callus growth from fescue embryos was much less than for the other two species and maximum callus growth with 2,4,5-T was only about one-half that obtained with 2,4-D.

Meiotic analyses of tall fescue plants regenerated from callus cultures

Abstract Meiotic analyses were conducted on tall fescue ( Festuca arundiancea Schreb). plants, (1) grown from seed, (2) regenerated from callus cultures and (3) regenerated from callus cultures in which the explant (mature embryos) had been previously treated with ethylmethane sulfonate (EMS). No choromosomal abnormalities were found in pollen mother cells of control plants. Between 40 and 50% of plants regenerated from tissue cultures exhibited abnormalities, including aneuploidy. Regenerated plants from treated and nontreated embryos did not differ appreciably in either frequency or types of aberrations observed. The most common aberrations were lagging chromosomes, bridges and micronuclei in dyads and tetrads. Pollen stainability was high in all three groups of plants and was not related to frequency of aberrations in individual plants.

The influence of osmotic pretreatment and inoculum age on the initiation and regenerability of switchgrass suspension cultures

SummaryExperiments were conducted to study the influence of osmotic pretreatment and inoculum (callus) age on the initiation and induction of somatic embryogenesis from suspension cultures of ‘Alamo’ switchgrass (Panicum virgatum L.). Embryogenic 10-, 20-, or 30-d-old calluses (hereafter referred to as inocula), produced from in vitro-cultured inflorescences, were used as explants to initiate the suspensions. Inocula were cultured for 30 h on MS solid medium containing 0.1, 0.2 or 0.3 M each of sorbitol and mannitol as an osmotic pretreatment. They were then transferred to liquid MS medium with 5 µM 6-benzylaminopurine and 9 µM 2,4-dichlorophenoxyacetic acid and cultured for 28 d in 2-ml Multiwell™ plantes. Individual multiwell contents were transferred to 125-ml Erlenmeyer flasks containing 20 ml of the above liquid medium and cultured for an additional 2 wk. Cultures initiated from the 10-d-old inoculum produced a higher embryogenic response than those initiated from 20- or 30-d-old inocula. Cultures initiated with 30-d-old inocula produced nonembryogenic clusters and the number of embryogenic cells was low. More embryos and a higher regeneration frequency were produced by 0.3 M than by 0.1 or 0.2 M of each of the osmostica.

Proportions of 2C and 4C nuclei in the root and shoot of dormant and germinated embryos of Festuca arundinacea and Dactylis glomerata

Abstract The 2C and 4C nuclear DNA content in the root and shoot of both dormant and germinated (72 hr at 20°C) embryos of Festuca arundinacea and Dactylis glomerata was determined by Feulgen microspectrophotometry. A cold hydrolysis technique was employed, and squashes were made of roots and shoots separately. Results showed predominantly 2C nuclei in both the root and shoot of dormant embryos of both species suggesting that the cells were arrested in G1 during embryo maturation. A mixed population of cells was observed in both roots and shoots of germinated embryos of both species. The proportion of 2C, S, and 4C nuclei were approximately the same in the root versus the shoot of Festuca; whereas, in Dactylis, a higher proportion of 4C nuclei and a lower proportion of 2C nuclei were observed in the root compared to the shoot.

Using pressure-volume analysis to characterize NaCl-induced osmotic and turgor adjustment in in vitro cultures.

Summary The applicability of pressure-volume (PV) analysis for characterizing osmotic and turgor adjustments in in vitro cultures was tested using salinized orchardgrass callus. Thermocouple psychrometry was used to derive PV curves in three experiments: (a) adjustment to varying concentrations of NaCl, (b) recovery from NaCl stress and (c) influence of NaCl acclimation to an additional, increased NaCl challenge. Callus growth and water relations were unaffected by 50 mM NaCl. Harvest and zero turgor osmotic potentials were decreased about 0.7 and 0.9 MPa, respectively, in callus grown for three weeks on 100 and 150 mM NaCl. Turgor was 0.2 to 0.4 MPa higher in 100 and 150 mM treatments across a range of callus water potential, compared to 0 or 50 mM NaCl. Salinization resulted in decreased estimates of callus elasticity and increased values of relative water and osmotic water content at zero turgor. The effects of NaCl stress on elasticity and osmotic potential were still evident three weeks after transfer to fresh medium containing 0 mM NaCl. A three-week acclimation of callus to 50 mM NaCl increased turgor and decreased osmotic potential and elasticity compared with unacclimated callus when cultures were subsequently grown on 100 mM NaCl. Apoplastic water percentages ranged from 21 to 35 % and showed no response to salinization. Psychrometric PV analysis appears to offer a promising alternative for the assay of component water potentials and water contents, elasticity and apoplastic/symplastic water partitioning in callus tissue.

A discussion on potential in vitro applications for agronomic crops

Abstract The round-table discussion on in vitro manipulations with agronomic crops was directed toward potential applications of importance in the improvement of agronomic crops. Emphasis was centered on species in the Gramineae and Leguminosae families. Topics for discussion included: (1) clonal propagation, (2) meristem tip culture, (3) induction and selection of mutants, (4) generation and use of haploids and (5) wide hybridization and protoplast culture. Potential applications and problem areas under each of these areas were identified and discussed. A major problem remains one of inability or low efficiency of plantlet regeneration from cells and tissues of economically important species in both of the above families. This topic received much discussion and possible research areas were suggested. The potential problems and uses of genetic and cytogenetic instabilities arising from in vitro cultures were also discussed extensively. Although in vitro culture techniques of important agronomic crops lag behind that of certain other species, optimism was expressed for future progress in this area.