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1

Isolated microspores as a source doubled haploids of barley

100%
Oleszczuk S., Zimny J.
Biotechnologia
|
2001
|
issue 1
139-142
EN
In vitro techniques for doubled haploids (DH) production allow for obtaining homozygous lines in a single generation. This is connected with shorter breeding cycle of the new variety. DH lines have a potential for being used in the selection of recombinants, stabilising of transformed lines and molecular mapping. DH lines are produced from isolated microspores through haploid embryogenesis. Microspore culture has several advantages over anther culture: it reduces the time of cultures, enables monitoring of the earliest phase of embryogenesis, allows for direct development embryos, facilitates the in vitro selection and mutation, allows for avoiding regeneration from somatic anther tissues. Moreover, microspore culture appears to be a promising tool in genetic manipulations (transformation, mutagenesis) and it can be used as a source of protoplasts and suspensions. Here we report on how to induce microspore embryogenesis, resulting in plant formation. The switch of microspore development from gametophytic to sporophytic pathway has been stimulated by various stress factors like cold and heat shock, starvation. Stress treatment not only stops pollen development but also re-programmes the microspore towards embryo formation. The effects of various parameters including pretreatment, carbohydrates and nurse culture have been investigated. After optimising the culture conditions we were able to regenerate high number of fertile plants.
2

In vitro culture of cereal microspores

100%
Oleszczuk S., Zimny J.
Biotechnologia
|
2000
|
issue 2
142-160
EN
The development of microspore culture methods in the Poaceae family has received considerable attention in recent years. Isolated microspore culture can be induced in vitro to switch their development from gametophytic to a sporophytic patway, resulting in embryoid or callus formation. Different stresses like cold or heat shock and nitrogen starvation have been identified as the major trigger in inducing microspore embryogenesis. Microspore culture appears to be a promising toll for future production of double haploids in cereals. Isolated microspore culture has several advantages over anther culture in genetic manipulation and haploid study, such as: direct observation of microspore development, unique possibility to study plant embryogenesis, easier in vitro selection and mutation, easier transformation of single cells. Moreover, isolated microspores are the most efficient way of double haploid regeneration. Many factors such as genotypes, physiological status of donor plants, stage of microspores development, pretreatment of anthers or spikes, method of microspores isolation, culture media, nurse culture and culture conditions, have a great influence on microspore culture. These and other problems concerning in vitro culture of isolated microspore are discussed in this review.
3

In vitro regeneration and transformation of rye (Secale cereale L.)

81%
Sowa S., Sowa A., Zimny J.
Biotechnologia
|
2000
|
issue 4
88-92
EN
In vitro cultures are an integral part of plant transformation. Genetic manipulation can be performed only on a single cell level. Therefore in vitro culture and regeneration of plants from a single cell are very important for successful transformation In vitro culture of rye is more difficult to conduct than of others cereals. Difficulties with in vitro regeneration of rye seems to be the main factor limiting the development of rye transformation systems. Genetic transformation process includes three main steps, single cell transformation, selection of transgenic cells and regeneration of plants from single cells. Efficiency of each of these steps can influence the result of the transformation process. Therefore optimisation of those steps is very important.Transformation has been performed using the microprojecticle method and scutellum of rye embryos as a target. Two constructs have been used in cotransformation, pDB1 containing a marker bar gene (phosphinotricine acetylotransferase) for Basta herbicide resistance and a repoter uidA gen (beta-glucuronidase) and pAwact-Sec containing 196 bp fragment of a sec-1 gene in anti-sense orientation. Using pAWact-Sec construct we tried to block the expression of the endogenous gene sec-1 coding omega-secalin which is storage protein of rye grain. We were able to regenerate transgenic rye plants containing all introduced genes. The efficiency of sec-1 expression blocking was analysed by SDS-PAGE method. Among 50 analysed kernels of T1 generation we found 5 with lower omega-secalin level. Complete blocking of omega-secalin was not observed.
4

The influence of various light spectra on the efficiency of somatic embryogenesis

81%
Menke-Milczarek I., Zebrowski J., Zimny J.
Biotechnologia
|
2001
|
issue 2
99-103
EN
The aim of presented work was to investigate the impact of various light spectra on the efficiency and intensity of wheat somatic embryogenesis. The efficiency of somatic embryogenesis was defined as a percentage of explants forming somatic embryos in reference to all cultured explants. Immature embryos at the spherical coleoptile stage were excised from seeds of a few varieties of wheat and placed onto MS medium (1962) supplemented with 30 muM Dicamba. The influence of blue, white and red light on the callus growth and induction of somatic embryogenesis was compared. Increase in proportion between the red (600-700nm) and blue (400-500nm) component of light spectrum accelerated development of somatic embryos and increased the efficiency of somatic embryogenesis.
5

Instability of ploidy level during regeneration of transformed and non-transformed tobacco (Nicotiana tabacum L.)

81%
Sliwinska E., Zimny J., Drozdowska J.
Biotechnologia
|
2003
|
issue 3
260-266
EN
In plant tissue cultures, somaclonal variation is often observed. It can be an effect of the changes in the individual chromosome number or in the ploidy level. Flow cytometry, a fast and accurate method for the estimation of the nuclear DNA content, can be applied to study these changes. The DNA content in differentiated tissues of Nicotiana tabacum cultured in vitro was estimated using Partec CCA flow cytometer, starting from explant, through callus, up to regenerated shoots. The explant constituted stem segments of N. tabacum plants, non-transformed and transformed with gfp gene. Flow cytometric analysis showed differences in the proportion of 2C, 4C, 8C and 16C cells in plant tissue in different culture stages. Among the regenerated plantlets originated from non-transformed and transformed plants, diploid, tetraploid and mixoploid forms were observed. The transformation did not influence the share of cells representing different ploidy levels in the investigated plant material.
6

Polish transgenic cereal crops

71%
Zimny J., Sowa S., Menke-Milczarek I., Czaplicki A., Sowa A.
Biotechnologia
|
2000
|
issue 4
80-87
EN
First transgenic cereal plants have been obtained in Poland seven years ago. Within the time other cereals like wheat, rye and barley have been also transformed. The prerequisite for that was a very efficient regeneration system by somatic embryogenesis. Generally the basic study on transgenic cereals are quite advanced but the question is how to include transgenic lines in to practical breeding process? Most of genes, promoters and transformation methods are patented and probably Polish breeders will never afford to buy the licences. Though there is a need to concentrate the future work in Polish institutes on identification and isolation of genes of interest. Than to transfer them to plants and register transgenic varieties. According to the Polish law it is allowed to carry out the field experiments, but it is not possible to register the plant variety.
7

Consequences of transformation of embryogenic Triticale cell

71%
Zimny J., Czaplicki A., Menke-Milczarek I., Oleszczuk S., Sowa S.
Biotechnologia
|
2000
|
issue 2
64-72
EN
Plants carrying foreign genes have been obtained for many crops including wheat, rice, maize, barley and Triticale. The most important aspect for practical breeding is the regeneration of whole plants from a specific cell possessing the desired agronomic properties. Particle bombardment provided the necessary breakthrough for the efficient transformation of cereals. Efficient regeneration is a prerequisite for all transformation techniques. The aim of the presented work was to study the progeny of transgenic plants of the allohexaploid cereal species Triticale. By combining an efficient regeneration system with the successful particle bombardment method we were able to obtain transgenic Triticale plants. Transgene expression was sometimes unstable and generally resulted in the decline of the expression, although some lines showing stable expression were also selected. In our laboratory several generations of androgenic doublehaploid transgenic lines have been regenerated and multiplicated. The integrated transgenes were detected in Triticale lines by in situ hybridisation method. The stability of trangenes has been studied on ten generations. A regeneration system from single cell to plant combined with microprojectile bombardment appeared to be the most efficient transformation method for Triticale. Numerous chimeric genes are now available for research. Some of these genes may appear useful in the future breeding of Triticale.
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