Shyamala Bhaskaran

Somatic embryogenesis from shoot tip and immature inflorescence of Phoenix dactylifera cv. Barhee

SummaryA method of clonal propagation via somatic embryogenesis of date palm, cultivar Barhee, which has potential for large scale commercial application as well as for developmental studies on embryos is described. Cultures were initiated from shoot tip and immature inflorescence explants, both of which were capable of development into embryogenic callus. When the embryogenic callus was cultured in liquid suspension on a rotary shaker, hundreds of embryos developed from milligram quantities of callus in a fairly synchronous manner. Scanning electron microscopy showed globular, heart-shaped and torpedo-shaped embryos. Green leaves emerged from a white cotyledonary sheath.

Enhanced somatic embryogenesis in Sorghum bicolor from shoot tip culture

SummaryShoot tip cultures from 2- to 3-d-old seedlings ofSorghum bicolor (L.) Moench cv. IS3620C develop highly embryogenic callus from which plants can be regenerated when transferred to plant growth regulator-free medium. Isolated shoot tips were cultured on Murashige and Skoog medium supplemented with 2.5 mg/liter 2,4-dichloro-phenoxyacetic acid and 0.05 mg/liter kinetin. Purple pigmentation characteristic of sorghum cultures on growth regulator-free medium is virtually eliminated with the shoot tip culture. Embryogenic callus is white and hard with an undulating appearance but can be separated into multiple bipolar structures by application of gentle pressure. The well-developed embryos have a cup-shaped scutellum. These germinate like zygotic embryos and develop root-shoot axis. Lack of vascular connections to the parent tissue and the synchronous development of the plumule and radicle suggest that these embryos may be of unicellular origin.In contrast, when the entire seedling serves as the explant, all meristematic centers in the shoot, including the coleoptile sheath close to the apical meristem respond to plant growth regulators in the medium by callus formation. Upon subsequent reculture onto growth regulator-free medium several modes of development occur. The differential response of these tissues to identical culture conditions indicate the presence of different population of cells that respond differently to exogenous plant growth regulators.

Sorghum plant regeneration from aluminum selection media.

SummaryCallus cultures of four Sorghum bicolor (L.) Moench cultivars were initiated from aseptically germinated seed. Prior to culture on media containing 0, 100, 200, and 400 μM aluminum, the callus had been subcultured for 3 to 12 months. There were differences amongst the cultivars in response to the aluminum in terms of callus growth. Growth was inhibited at the highest level of aluminum tested. Subculture of callus from aluminum selection medium on both aluminum-containing and aluminum-free media indicated that aluminum-selected callus grew better in the presence of aluminum. Plants were regenerated from only one cultivar (IS3620C of the Margaritiferum group) after several passages on aluminum-supplemented media. Regeneration capacity was however inhibited at 400 μM aluminum.

Somaclonal variation from Sorghum bicolor (L.) Moench cell culture

Sorghum bicolor (L.) Moench, plants were regenerated from 4 to 5 month old callus cultures originally derived from seedling explants. Somaclonal variation was examined in the SC2 and SC3 generations of eight different clones plus the non-tissue cultured parent line. Characters examined included the area of the third leaf, height, tiller number, total shoot weight, seed number, grain yield, days to flowering, and chlorophyll content in the SC2 generation. Some plants were phenotypically different from the parent line in the SC2 generation. All clones were similar to the parent in third leaf area and chlorophyll content. Three clones had significant height reduction and six had significantly higher total plant matter production. Seven of the clones had significant increases in number of secondary tillers, and all had significant decreases in days to flowering. Only three clones had significant increases in grain yield and seed number; however, all the clones had smaller seeds. In subsequent studies of SC2 and SC3 generations of three somaclones, characters examined included height, tiller number, total shoot weight, and days to flower. In one of the somaclones none of the original differences in characters was maintained in two subsequent growing seasons in the SC2 or SC3 generation. The other two somaclones maintained the increased tiller number in the next growing season in both the SC2 and SC3 generations. In one of the somaclones plant height reduction occurred in the SC2 and SC3 generations. All other traits were not maintained.

Sodium Chloride Tolerant Callus of Sorghum bicolor (L.) Moench.

Summary Callus cultures of Sorghum bicolor (L.) Moench cv. Margaritiferum obtained by prolonged culture on NaCl-containing medium were able to grow better on NaCl-containing medium than the parent cells. After every passage on the stress selection medium, part of the cultures were transferred to hormone-free medium for plant regeneration. Plants were regenerated from calli grown on NaCl-containing media for up to five passages. Some of these plants were albinos. A few green plants have been grown to maturity in the greenhouse. There was very low seed set in these plants. Attempts are underway to regenerate more plants from stress selection media and also to get better seed set from the plants obtained.

Somatic embryogenesis in date palm (Phoenix dactylifera L.)

Phoenix dactylifera L., date palm, origins date back 4,000 to 5,000 years ago and are evident in the construction of a temple near Ur in Iraq (FAO, 1982). Current distribution of date palm is approximately 20° N and 35° N including dry, subtropical climates in northern and southern hemispheres of North and South America, Africa, and Asia. Recently it has been introduced into the Colorado Desert of North America, the Atacama Desert of South America, and the Kalahari Desert of South Africa, and into the Central Desert of Australia. The date palm requires hot summers without rain or high humidity for the five to seven months from pollination to harvest. Irrigation, however, is necessary.

Progeny Screening of Sorghum Plants Regenerated from Sodium chloride-selected Callus for Salt Tolerance

Summary Sorghum bicolor (L.) Moench plants regenerated from callus cultures grown with and without salt (NaCl) were grown to maturity in a greenhouse. Seeds collected from these populations after self-pollination were germinated, and seedlings were screened in a hydroponics system for NaCl tolerance. Seedlings from the parent plants also were included in the screening. Progeny from the salt-selected plant had slightly longer roots than the parents when grown in normal nutrient solution. In nutrient solution containing NaCl, the salt-selected progeny had higher shoot dry matter than did the parent line. However, progenies from both the parent and salt-selected lines had decreased root weights and root lengths in the presence of NaCl. This suggests that seedlings from the salt-selected plant obtained adequate nutrients to allow higher shoot dry matter accumulation than that of the parent population.

Screening for drought tolerance in Sorghum using cell culture

SummaryCallus growth from 10 cultivars ofSorghum bicolor (L.) Moench was measured with increasing levels of polyethylene glycol (PEG) as an osmoticum in the medium to determine whether differences among these cultivars at the cellular level in response to osmotic stress existed. These cellular ratings were compared to field ratings from the 10 tolerant-to-susceptible cultivars when grown under drought conditions to determine whether cellular ratings corresponded to differences in drought tolerance at the plant level. Callus cultures were grown on Murashige and Skoog inorganic salt formulation plus vitamins, 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin and sucrose, supplemented with 0 to 25% (wt/vol) PEG corresponding to −0.2 to −1.62 MPa osmotic potential. Results suggest that PEG-induced osmotic stress on callus cultures can be used to screen sorghum cultivars for potential early field (preflowering) drought tolerance. This implies that at least a component of the early field drought tolerance in sorghum may have a cellular basis.

Origin of somatic embryos from cultured shoot tips ofSorghum bicolor (L.) moench

SummaryHistologic examination of shoot-tip explants, 1 wk after culture initiation on Murashige and Skoog medium with 2.5 mg/liter 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.05 mg/liter kinetin, reveals active meristematic centers inside cultured tissue. Clusters of cells in these meristematic centers exhibit remarkable resemblance to the initial three divisions in the zygotic embryo. Several such meristematic groups of cells are observed in the cultured explant at this stage. Embryogenesis is obviously initiated very early in this tissue in the presence of 2,4-D. A well-defined, white globular embryogenic callus develops in culture in about 4 wk, and it consists of clusters of embryoids with large cells characterized by thick cell walls, numerous lipoidal vesicles, and localized areas of carbohydrate storage. These cells resemble the scutellar tissue of the embryo. However, there are cells within this tissue that themselves appear embryogenic. They undergo cell division giving rise to small clusters of cells. As long as 2,4-D is present in the medium, the cells apparently retain the capacity to proliferate and to produce more cells capable of embryogenesis. Embryogenesis seems to occur via two processes, initiation of somatic embryos early in culture and secondary embryogensis from the scutellar tissue that forms in vitro.

Carbohydrates, invertase activity, growth and dimorphism inSporisorium reilianum

Sporisorium reilianum, the fungus that causes sorghum head smut, was grown with sucrose, lactose, trehalose or raffinose in liquid suspension or on a solid medium. Liquid culture media were analyzed for hydrolysis products of these carbohydrates to determine extracellular enzyme activity of the fungus. Increased amounts of glucose and fructose in the culture medium ofS. reilianum grown with sucrose or raffinose indicated that invertase (β-fructofuranosidase, 3.2.1.26) activity was present. No evidence of extracellular galactosidase or trehalase activity was found. Enhanced sporidial colony formation on carbohydrates that can be hydrolyzed to hexoses, and specific forms of mycelial growth on lactose, trehalose or on a carbohydrate-deficient medium might suggest that mycelial growth is a way of foraging for food sources. However, the rapid and profuse mycelial growth on the host cell wall glycoprotein appears to be in response to abundant food supply (probably of a different type). Therefore availability of different kinds of carbon sources in the environment of the growing fungus might determine dimorphism and associated pathogenesis byS. reilianum.