Kutty K. Kartha

Induction of Somatic Embryos and Plantlets from Cryopreserved Cell Cultures of White Spruce (Picea glauca)

Summary A cell suspension culture of Picea glauca with potential for sustained somatic embryo production has been successfully cryopreserved retaining very high levels of viability even after 1 year storage in liquid nitrogen. The following cryobiological components were identified as optimal for the recovery of viable cells postcryopreservation: 1) preculturing the cells for 24 h prior to freezing, in liquid nutrient medium enriched with 0.4 M sorbitol, 2) treating the cells after preculture with a cryoprotectant combination of 0.4 M sorbitol + 5 % DMSO, and 3) controlled freezing at a cooling rate of 0.3 °C min −1 to −35 °C followed by storage in liquid nitrogen. Dimethylsulfoxide alone at concentrations ranging from 5 to 20 %, in addition to being cytotoxic, was ineffective as a cryoprotectant. The callus re-established from cryopreserved cells maintained embryogenic potential identical to the unfrozen controls. Light and scanning electron microscopic examination of somatic embryos revealed structural and organizational conformity with those induced from unfrozen controls. Calli regrown from cryopreserved cells were induced to differentiate into plantlets through embryogenesis, and into shoots by organogenesis.

Genetic transformation of strawberry by Agrobacterium tumefaciens using a leaf disk regeneration system.

An efficient genetic transformation protocol has been developed for strawberry cv. Redcoat using Agrobacterium tumefadens. The protocol relies on a high frequency (84%) shoot regeneration system from leaf disks. The leaf disks were inoculated with a non-oncogenic Agrobacterium tumefadens strain MP90 carrying a binary vector plasmid pBI121 which contains a chimeric nopaline synthase (NOS) promoter driven neomycin phosphotransferase (NPT II) gene and a cauliflower mosaic virus 35S (CaMV35S) promoter driven, ß-glucuronidase (GUS) marker gene. The inoculated leaf disks, pre-cultured for 10 days on non-selective shoot regeneration medium, formed light green meristematic regions on selection medium containing 50 μg/ml kanamycin. These meristematic regions developed into transformed shoots at a frequency of 6.5% on a second selection medium containing 25 μg/ml kanamycin. The selected shoots were multiplied on shoot proliferation medium in the presence of kanamycin. All such shoots were resistant to kanamycin and expressed varying levels of NPT II and GUS enzyme activity. Histochemical assays for GUS activity indicated that the 35S promoter was highly active in meristematic cells of shoot and root apices. Molecular analysis of each transgenic clone confirmed the integration of both marker genes into the strawberry genome. Leaf disks prepared from transformed plants, when put through the second selection cycle on kanamycin, formed callus and exhibited GUS activity. The rooted transformed plants were grown in a greenhouse for further characterization. The protocol may be useful for improvement of strawberry through gene manipulations.

Comparison of free sugars in growing and desiccated plants of Selaginella lepidophylla

Abstract The free sugars are growing and desiccated Selaginella lepidophylla plants were examined. Growing and desiccated plants differed significantly in their percentage of glucose (3.1, 0.2%), sucrose (6.9, 23.1%) and trehalose (89.8, 75.6%). The apparent stoichiometry between the decrease in sucrose and increase in trehalose concentration after rehydration suggests that sucrose serves as the carbon source for trehalose synthesis. A comparison of extraction methods revealed that using “Boileezers” in Soxhlets resulted in sugar degradation ranging from 25 to 75%. Extraction by shaking in 80% ethanol appeared to be the best extraction method.

Transient expression of chloramphenicol acetyltransferase (CAT) gene in barley cell cultures and immature embryos through microprojectile bombardment

Transient expression of chloramphenicol acetyl transferase gene has been detected in cultured barley (Hordeum vulgare L. cv. Heartland) cells and freshly isolated immature zygotic embryos (cv. Ellice) following the introduction of the gene by microprojectile bombardment. The DNA expression vector used to introduce the CAT gene, pCaMVI1CN, is a pUC8 derivative and consisted of a CaMV35S promoter, a fragment of alcohol dehydrogenase intron1, a CAT coding region and NOS polyadenylation region. The inclusion of the Adh1 intron1 was essential for the expression of CAT activity in cultured cells as well as immature zygotic embryos. Expression of CAT activity, which was dependent upon the DNA concentration used, could be detected as early as 20 h after bombardment. The results also suggested that the recipient cells have to be in an active state of cell division in order for the introduced gene to be expressed since mature zygotic as well as somatic embryos failed to reveal any gene expression. The effect of other parameters which influence the expression of the introduced gene as well as the potential of this novel technology for cereal transformation are also discussed.

Improved embryoid induction and green shoot regeneration from wheat anthers cultured in medium with maltose.

SummaryAnthers from spring wheat (Triticum aestivum L.) genotypes, including six F1 hybrids, were cultured in a modified liquid N6 medium containing either sucrose or maltose. In every case, use of maltose resulted in greater microspore callus induction and green shoot regeneration than culture in sucrose-containing medium. Induction in maltose medium also allowed green shoots to be recovered from crosses that showed only a poor response in other media and from two genotypes that did not respond to modified N6 medium with sucrose. Replacement of sucrose with maltose generally resulted in microspores having a more embryogenic mode of development in which distinct embryoids often formed. The most responsive genotype produced over 200 green shoots/100 anthers when cultured in medium with maltose.

Agrobacterium-mediated transformation of strawberry calli and recovery of transgenic plants

Transformed calli and shoots of strawberry (Fragaria × ananassa Duch.) cv. Redcoat were obtained using Agrobacterium tumefaciens carrying plasmid pB1121. Inoculated leaf explants produced transgenic calli at a frequency of 3% on selection medium containing 50 μg/ml kanamycin. Twenty per cent of selected caili regenerated, giving rise to transgenic shoots. All transgenic calli and shoots expressed substantial amounts of GUS and NPT-II activity. The Southern blot analysis confirmed the insertion of both marker genes into the strawberry genome as single and multiple copy inserts. The transgenic shoots elongated on rooting medium in the presence of 25 μg/ml kanamycin, but exhibited reduced rooting ability.

The influence of plant growth regulator concentrations and callus age on somaclonal variation in callus culture regenerants of strawberry

The effect of plant growth regulator concentrations and ageing of callus on the extent and nature of variation among callus culture regenerants of strawberry (Fragaria × ananassa) cv. Redcoat was examined. Plants regenerated from callus culture had reduced plant vigour, shorter petiole length and smaller leaf size, but more leaves and runners under greenhouse conditions. These responses appeared to be due to a physiological influence of plant growth regulators. No distinct phenotypic variants were observed at plant growth regulator concentrations in the range of 1–10 μM each of BA and 2,4-d combination, but the highest concentration (20 μM each) of this combination produced a high frequency (10%) of dwarf type variants. The dwarf nature of these variants was maintained in the runner plants produced by the primary regenerants. The plants regenerated from 8-week-old calli did not show any distinct morphological variants. However, a significant proportion of deformed leaf shape (6–13%) and yellow leaf (21–29%) variants was obtained among plants regenerated from 16 and 24-week-old calli. The primary regenerants of the leaf shape variants were established as chimeras. The chimeric plants produced runner progeny with normal plants and plants with completely distorted leaf morphology. Both leaf shape and yellow leaf variants remained stable through runner propagation. Isozyme analysis failed to distinguish any of the variants from the standard runner plants. Flow cytometric analysis indicated the aneuploid nature of leaf shape variants but it could not distinguish dwarf and yellow leaf variants from standard runner plants.

In vitro Growth Responses and Plant Regeneration from Cryopreserved Meristems of Cassava (Manihot esculenta Crantz)

Summary A droplet-freezing method has been developed for the cryopreservation of cassava, Manihot esculenta Crantz, meristems. The meristems treated with a 15 % DMSO + 3% sucrose solution for 15 min were frozen over an 18 μm aluminium foil in 2–3 μl droplets of the cryoprotectants in plastic petri dishes at a cooling rate of 0.5°C/min to various sub-zero temperatures (-20; -25; -30 and -40°C) and stored in liquid nitrogen (-196°C). The meristems retrieved from liquid nitrogen storage, upon thawing and return to in vitro culture on plant regeneration medium, exhibited various morphogenetic responses such as differentiation of callus and leaves and whole plantlets. The plantlets were successfully grown in pots.

Immature embryo and anther culture of chromosome addition lines of rye in chinese spring wheat

Abstract Significant variation among Chinese Spring wheat ( Triticum aestivum L.) and a set of seven addition lines in which chromosomes from rye ( Secale cereale L.) were incorporated into the Chinese Spring background was observed for callus formation and plant regeneration from anther cultures and for plant regeneration from immature embryo cultures. Callus initiation from immature embryo cultures was uniformly high. Rye chromosome 4 contains factors which significantly increase both anther culture responses relative to Chinese Spring. Rye chromosomes 6 and 7 both contain positive factors for regeneration from immature embryo culture. While no correlation was found between anther culture and embryo culture responses, a positive correlation was observed between the two anther culture response variables.

Cryopreservation of wheat suspension culture and regenerable callus

Wheat (Triticum aestivum L. cv. Norstar) suspension cultures and regenerable calli initiated from immature embryos can be cryopreserved in liquid nitrogen temperature (−196°C) by slow freezing (0.5°C/min) in the presence of a mixture of DMSO and sucrose or sorbitol. Cold hardening or ABA treatment before cryopreservation increased the freezing resistance and improved the survival of wheat suspension culture in liquid nitrogen. Callus culture, established from immature embryos, prefrozen in 5% DMSO and 0.5M sorbitol survived liquid nitrogen storage and resumed plant regeneration after thawing. The results confirm the feasibility of long term preservation of wheat embryo callus by cryopreservation and retention of plant regeneration ability.