Maureen M. M. Fitch

High-density linkage mapping revealed suppression of recombination at the sex determination locus in papaya.

A high-density genetic map of papaya (Carica papaya L.) was constructed using 54 F2 plants derived from cultivars Kapoho and SunUp with 1501 markers, including 1498 amplified fragment length polymorphism (AFLP) markers, the papaya ringspot virus coat protein marker, morphological sex type, and fruit flesh color. These markers were mapped into 12 linkage groups at a LOD score of 5.0 and recombination frequency of 0.25. The 12 major linkage groups covered a total length of 3294.2 cM, with an average distance of 2.2 cM between adjacent markers. This map revealed severe suppression of recombination around the sex determination locus with a total of 225 markers cosegregating with sex types. The cytosine bases were highly methylated in this region on the basis of the distribution of methylation-sensitive and -insensitive markers. This high-density genetic map is essential for cloning of specific genes of interest such as the sex determination gene and for the integration of genetic and physical maps of papaya.

Pathogen-derived resistance provides papaya with effective protection against papaya ringspot virus

Transgenic Carica papaya plants (cv. Sunset, R0 clone 55-1) carrying the coat protein gene of papaya ringspot virus (strain HA 5-1) remained symptomless and ELISA-negative for 24 months after inoculation with Hawaiian strains of papaya ringspot virus under field conditions. Non-transgenic and transgenic control plants lacking the coat protein gene developed disease symptoms within one month after manual inoculation or within four months when natural aphid populations were the inoculum vectors. Mean trunk diameter was significantly greater in cloned 55-1 plants compared with virus-infected controls (14.7 cm versus 9.3 cm after 18 months). Fruit brix, plant morphology, and fertility of 55-1 plants were all normal, and no pleiotropic effects of the coat protein gene were observed. These results indicate that pathogen-derived resistance can provide effective protection against a viral disease over a significant portion of the crop cycle of a perennial species.

Long-term culture of embryogenic sugarcane callus

Embryogenic calluses of sugarcane capable of regenerating green plants after long-term culture were sought. The largest quantities of embryogenic calluses were produced on Murashige & Skoog medium, but cultures maintained on Chu N6 medium remained embryogenic and totipotent longer. Both media contained 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-d). The effect of supplements on somatic embryogenesis was examined. Kinetin (0.5 μM) and 10% (v/v) coconut water in callus initiation medium were inhibitory to subsequent embryogenesis. Embryogenic calluses on N6 medium increased in fresh weight with proline concentration up to 90 mM. Maximum fresh weight was achieved with 5% sucrose. Although genotypic differences were observed, embryogenesis occurred in all 17 sugarcane clones tested. Embryogenic calluses of one cultivar regenerated green plants after 16 months, but suspensions were totipotent for only 8 months. Total number of regenerated plants decreased with time in culture, while the number of pale green plants increased starting after 5 months in culture.

Haploid Production From Anther Culture of Saccharum spontaneum L.

Summary Initiation of microspore multicellular development in anther cultures of Saccharum spontaneum L. was stimulated by preculture treatment of panicle branches at 10°C for 21 d. This prolonged cold treatment killed young microspores and switched the development of nearly mature microspores from a gametic to a sporophytic route. Anther cultures produced microspore-derived calli in four of the ten clones tested. Calli of two clones yielded plants; one line was haploid, the other diploid.

The induction of callus in sugareane tissue cultures by selected chemicals

Immature leaf tissue of two commercial clones of sugarcane was subjected to tissue culture in Louisiana and Hawaii. The callus-incuding activity of selected compounds at 2, 4, and 20 mg/l was compared to the activity of 2,4-D at the same concentrations and to tissue on medium without a callus-inducing agent. Of the 79 compounds tested, 25 induced callus and 54 were ineffective. Ninety-six percent of the effective compounds were in chemical groups with known auxin activity. The results suggested that simple ring structure, phenol derivatives, or a halogenated hydrocarbon chain are not sufficient to induce callus. When callus was transferred to a medium lacking the inductive chemical, differentiation into shoots, roots, or both occurred in callus produced by 80% of the effective compounds.

Production of Haploid Saccharum spontaneum L. - Comparison of Media for Cold Incubation of Panicle Branches and for Float Culture of Anthers

Means were sought to increase the frequency of haploid production from anther cultures of Saccharum spontaneum L. Excised preemerged panicle branches were incubated in modified Murashige-Skoog (MS), Nitsch (H), Gamborg (B5), and Chu (N6) media at 10 °C for 4 to 10 weeks prior to removal of the anthers for culturing at 27 °C. Anthers, either continuously cultured on liquid media or floated on liquid media 1 to 8 weeks prior to nurse culture, produced visible calli in 30 days. Nearly 1% of the anthers in one clone, SES 208, produced calli. The calli increased in mass on nurse medium containing 0.25 to 0.50 mg · l(-1) picloram as the auxin source and differentiated into green calli when the picloram was reduced to 0.05 to 0.125 mg · l(-1). The green calli developed into plants on lowered-picloram or picloram-free media. Calli and green plants were obtained from all three clones studied. Plants were regenerated from 100 out of 276 callus lines. Seventeen plant lines have been transferred to greenhouse culture. Four of seven plant lines checked for ploidy were haploid, two were diploid. A method utilizing stomate guard cell lengths was developed as a means to estimate ploidy level. The increased success in haploid callus and plant production is thought to be the result of long cold treatment of panicle branches, use of low salt media for panicle treatment in the cold, use of picloram in place of 2,4-D in the nurse culture, and use of a high nitrate/ammonium ratio in the liquid culture.

Culture of Isolated Microspores of Saccharum spontaneum)

Summary Microspore culture of Saccharum spontaneum L. was investigated as a means to produce haploid plants. Isolated and cultured microspores initiated sporophytic development which in 4 to 6 weeks resulted in procalli and calli. The percentage of microspores producing multicellular procalli was increased by removing the nonviable microspores before initiating the culture, establishing an optimum population density, and using nutrient media relatively low in sucrose and NH4NO3. The procalli produced from microspore culture ceased development at about the same stage as did procalli within anthers in culture. Thus, a factor or factors other than the anther wall must be responsible for the low yield of haploids from S. spontaneum. The effect of temperature and of sucrose and 2,4-D in the media changed during the development of calli from unicellular microspores. The apparently different requirements for specific stages of development indicates that successful production of haploid plants from microspore culture will require a series of environmental and cultural manipulations.

Haploids of sugarcane

Sugarcane is one of the world’s largest cultivated grasses and belongs to the family Poaceae. Sugarcane as a crop is grown in 106 tropical and subtropical countries (FAO, 1992). In 1992, the crop was grown on 1.8 x 10’ hectares yielding 1.1 x 109 metric tons (MT) of cane giving an average yield of 61 MT/ha. The crop is grown mainly for sucrose and for fiber and ethanol production. Sugarcane ranks second in production after cereals (2.0 x 109 MT), the cane tonnage being equivalent to that of wheat plus rice (0.5 and 0.6 x 109 MT, respectively).

Taro (Colocasia esculenta (L.) Schott)

Genetic engineering of taro is an effective method to improve taro quality and the resistance to various diseases of taro. Agrobacterium tumefaciens-mediated transformation of taro is more efficient than the particle bombardment transformation method based on current research. The development of a regeneration system starting from taro shoot tip explants could produce dasheen mosaic virus (DsMV)-free plantlets. Highly regenerative calluses could be developed from DsMV-free, in vitro plantlets on the Murashige and Skoog (MS) medium with 2 mg/L BA and 1 mg/L NAA (M5 medium). The Agrobacterium tumefaciens-mediated transformation method is reported in this chapter. The highly regenerative calluses were selected and cocultivated with the Agrobacterium strain EHA105 harboring the binary vector PBI121 with either a rice chitinase gene chi11 or a wheat oxalate oxidase gene gf2.8. After cocultivation for 3-4 days, these calluses were transferred to selection medium (M5 medium) containing 50 mg/L Geneticin G418 and grown for 3 months in the dark. Transgenic shoot lines could be induced and selected on the MS medium containing 4 mg/L BA (M15 medium) and 50 mg/L Geneticin G418 for 3 months further in the light. Molecular analyses are used to confirm the stable transformation and expression of the disease resistance gene chi11 or gf2.8. Pathologic bioassays could be used to demonstrate whether the transgenic plants had increased disease resistance to taro pathogens Sclerotium rolfsii or Phytophthora colocasiae.

Genetic Transformation of Taro

Taro (Colocasia esculenta (L.) Schott) is cultivated worldwide for its edible corms and leaves. It was the world’s fourteenth most-consumed vegetable and the fifth most-produced root crop in the world during 2010. However, various pests and diseases, especially fungal and oomycete diseases, are major problems causing steep declines in taro production. Conventional breeding of disease resistant cultivars is ongoing, although it is a lengthy process. Tissue culture and genetic transformation of taro are alternative options to improve yields, quality, and disease resistance. Compared with conventional breeding, genetic engineering has unique advantages, such as a much broader gene pool for selection of genes of interest and the capability of transferring only a few transgenes, thus maintaining all other desirable crop characteristics. Only a few reports are available on the regeneration and genetic transformation of taro. The first report of taro transformation described insertion of a reporter gus gene and a selection gene hpt into a Japanese taro cultivar via particle bombardment with a very low transformation efficiency. More recently, particle bombardment and Agrobacterium-mediated transformation methods have been used to transform a Chinese taro cultivar with a disease resistance gene chi11 from rice. The Agrobacterium-mediated method had much higher transformation efficiency than particle bombardment. Insertion of this rice chitinase gene into taro resulted in moderately increased disease resistance against the fungal pathogen Sclerotium rolfsii. These results demonstrate the potential usefulness of genetic transformation to increase disease resistance of taro, particularly in instances where there are no naturally occurring resistances within the taro germplasm or the elite taro cultivars are difficult to breed conventionally.