After a short introduction, the cytogenetics of plant cell and tissue cultures and their regenerates will be discussed. In the first section discussion will focus on cytogenetic conditions “in vivo”, i.e., in the original explant: (I) widespread ocurrence of polysomaty as a consequence of endoreduplication; (2) aneusomaty, an important, though rare, cause of chromosome number variation in vivo; (3) occurrence of chromosome structural changes in differentiated tissues, especially in association with aging; (4) mixoploidy and/or gene mutations, either nuclear or organellar, present as mosaics or periclinal chimeras, especially in vegetatively propagated plants. In section two the discussion will follow with nuclear processes at and during callus induction: (1) mitosis induction in diploid (haploid) and endoreduplicated cells and initiation of cell lines with different ploidy levels; (2) chromosome endoreduplication prior to mitosis induction as a mechanism of polyploidization; (3) nuclear fragmentation (ami...

Cytogenetics of plant cell and tissue cultures and their regenerates

Gene manipulation in plant improvement , Gene manipulation in plant improvement , مرکز فناوری اطلاعات و اطلاع رسانی کشاورزی

Gene manipulation in plant improvement

In recent years, the rapid development of somatic cell genetics has made possible the transfer of alien genes over wide taxonomic distances by somatic hybridization. In this review, the potential of somatic hybridization in the breeding of crops within the Brassicaceae, Fabaceae, Poaceae and Solanaceae is discussed. It is evident from these studies that many hybrids, either symmetric or asymmetric, which are fertile have the potential to be used as a bridge between the alien species and the crop. Progeny analysis of some hybrid combinations also reveals intergenomic translocations which may lead to the introgression of the alien genes. Furthermore, fusion techniques enable the resynthesis of allopolyploid crops to increase their genetic variability and to restore ploidy level and heterozygosity after breeding at reduced ploidy level in polyploid crops.

The potential of somatic hybridization in crop breeding

Publisher Summary Plant cells are surrounded by a complex cell wall composed primarily of polysaccharides. Techniques have been developed for the routine removal of this wall from a wide range of plant cells. The resulting naked cells or protoplasts provide a unique experimental system that is proving very useful for studying the structure and function of plant cells. Many manipulations that are not possible with intact plant cells are feasible with protoplasts. These include surface labeling of the plasma membrane, complete cell wall regeneration, and cell fusion. Protoplasts are particularly important for examining the plant plasma membrane, a component of the plant cell that is normally inaccessible because of the presence of a cell wall. This chapter examines the major applications of plant protoplasts to the study of plant cells. Emphasis is placed on structural studies of higher plant protoplasts especially by electron microscopic methods. Protoplasts are isolated from a wide variety of plant species and when cultured have produced callus and in some cases complete plants. This chapter presents several examples of plants that have been regenerated from protoplasts.

Applications of Protoplasts to the Study of Plant Cells

The fusion of gametic protoplasts with somatic protoplasts giving rise to gametosomatic hybrid plants was investigated. Gametosomatic hybrid plants were regenerated following the fusion of nitrate reductase deficient (Nr−) Nicotiana tabacum Nia-130 leaf mesophyll protoplasts with N. glutinosa tetrad protoplasts. The resulting plants were confirmed as hybrids, based on leaf and floral morphology, chromosome number, leaf esterase and leaf callus peroxidase zymograms and Fraction-1-protein analysis. The five gametosomatic hybrid plants had the expected pentaploid, but functionally triploid chromosome number of 3n=5x=60. The relevance of triploid gametosomatic hybrids in facilitating limited gene transfer, is discussed. The utilisation of tetrads as a generally available source of haploid protoplasts for fusion studies is proposed.

The production of fertile, triploid somatic hybrid plants (Nicotiana glutinosa (n) + N. tabacum (2n)) via gametic: somatic protoplast fusion.

Mesophyll protoplasts of Nicotiana tabacum. cv. Bright Yellow mutant Aurea and Burley 21, and of N. rustica. cv. Rustica were prepared from leaf tissue by enzymatic digestion and were fused with the aid of polyethylene glycol. When the fused protoplasts were plated on an agar medium or in a liquid medium for cell culture, new cell walls were regenerated from them and the first cell division could be seen beginning on the 12th day of culture, and cell division process took place on and after the 21th day (Fig. 1). After one month culture, almost all of the surviving fused protoplasts grew to cell colonies of 1-2 mm in diameter. The cell colonies were subcultured on an agar medium for callus culture. On the medium, they grew vigorouly and formed calli of 2 cm in diameter after 2 months culture. At this stage, the hybrid calli showing greenish white with a compact appearance were selected in contrast to parental type calli tinged with white or green color (Fig. 2). Ten somatic hybrid calli of the combination of Aurea and Rustica and 3 calli of the combination of Burley 21 and Rustica were obtained. Many plantlets were differentiated from these calli when transfered to a medium enriching zeatin (Fig. 3). All of the mature flowering plants showed to be perfectly hybrid, and the shape of the leaves and flowers indicated an intermediate between N. tabacum and N. rustica (Fig. 5, 6). The plant height of the normal hybrid morphologically was higher than that of the parent plant and all of the hybrid plants were highly resistant to the tobacco mosaic virus (Fig. 4, Table 1, 2). The chromosome numbers of the somatic hybrids showed to be the aneuploid type having from 60 to 91 chromosomes (Table 1, 2). The normal hybrid plants morphologically, however, have a pollen fertility, and seeds and progenies were obtained from 4 of the Aurea-Rustica hybrids and 3 of the Burley 21-Rustica hybrids (Table 1, 2).

Somatic Hybridization by Fusion of Protoplasts : 1. The combination of Nicotiana tabacum and Nicotiana rustica

Somatic Hybridization by Fusion of Protoplasts : III. Somatic hybrids of sexually incompatible combinations Nicotiana tabacum+Nicotiana repanda and Nicotiana tabacum+Salpiglossis sinuata

Mesophyll protoplasts of Nicotiana tabacum. cv. Bright Yellow mutant Aurea and N. glutinosa and of N. tabacum. cv. Burley 21 and N. alata were prepared from leaf tissue by enzymatic digestion and were fused with the aid of polycthylcne glycol. When the fused protoplasts were platcd on an agar medium or in a liquid medium for cell culture, new cell walls were regenerated from them and the cell division could be seen beginning on the 14th day of culture in the combination of N. tabacum and N. glutinosa, whereas in that of N. tabacum and N. alata 10th day of culture. Cell division process took place over again after that. After one month culture, almost all of survived protoplasts grew to cell colonies of 1-2 mm in diameter. The cell colonies were placed on an agar medium containing 3.0 mg/l l-Naphthalenacetic acid and 1.0 mg/l 6-Benzylaminopurine in order to induce callus formation. On the medium, they grew vigorously and formed calli of 2 cm in diametcr after 2 months culture. However, it was too difficult to select the hybrid callus. They were trans-fered to a modified LINSMAIER and SKOOG medium containing 1.0 mg/l kinetine and 1.0 mg/l indoleacetic acid. After one month of culture on this mcdium, the hybrid calli, showing greenish white with a compact appearance werc selectcd in contrast to the parental type calli tinged with a white or green color. Thirty-three somatic hybrid calli of the combination of N. tabacum and N. glutinosa and 2 calli of the combination of N. tabacum and N. alata were obtained. Many plantlets were differentiated from these calli when transfered to enriching zeatin. All of the mature flowering plants showed to be perfectly somatic hybrid, and the shape of the leaves and flowers indicated that many somatic hybrids were of an intermediate characteristic between N. tabacum and N. glutinosa and between N. tabacum and N. alata. A few plants had a strong resemblance to a parent or a scxual hybrid. The plant height of the many somatic hybrids were higher than that of the parental plants, and the male sterility of the greater part of the hybrid plants was visible under investigation, and it was possible to back cross. They were highly resistant to the tobacco mosaic virus. The chromosomc number of the somatic hybrids of the combination of N. tabacum and N. glutinosa was determined: fivc were amphidiploid, twenty-eight showed aneuploid with 50 to 88 chromosomes. On the other hand, thc chromosome number of the somatic hybrids of the combination of N. tabacum and N. alata also was determined : one was amphidiploid and the other aneuploid.

Somatic Hybridization by Fusion of Protoplasts : II. The combinations of Nicotiana tababum and N. glutinosa and of N. tabacum and N. alata

Influence of oxygen concentration in the culture medium on the translocation of assimilated 14CO2 into roots was studied on the tobacco seedling. The major results were as follows: 1. The translocation of 14C from top to root and its storage in the root was greater under aerobic condition than under anaerobic one. 2. The specific activity of 14C in the top scarcely decreased by the preceding anaerobic treatment, whereas that in the roots decreased conspicuously. 3. Exchange of anaerobic condition into aerobic one gave little influence on the specific activity in the top or decreased it to some extent, whereas that in the roots increased obviously.

Studies on the Growth of Tobacco Roots : IV. Effect of oxygen concentration of media on the translocation of 14C assimilated into root

The nicotine synthesis was examined in the big and fine root tips and in the roots varying in kind, ages and regions of roots, furthermore in the roots grown in the anaerobic and aerobic media. The results obtained were as follows. 1. Nicotine content in the roots of the class C and D whose length were 20-30 mm and 30-40 mm respectively which emerged from tobacco seedlings was higher than that in the other classes. 2. The nicotine content became higher with the increasing distance from growing tip. The root tips in the aerobic water medium produced larger quantities of nicotines than did the anaerobic one. 3. The content and synthesis of nicotine in the fine root tips of the several varieties were higher than in the big roots.

Teruyoshi Nagao

Papers

Somatic Hybridization by Fusion of Protoplasts : 1. The combination of Nicotiana tabacum and Nicotiana rustica

Somatic Hybridization by Fusion of Protoplasts : III. Somatic hybrids of sexually incompatible combinations Nicotiana tabacum+Nicotiana repanda and Nicotiana tabacum+Salpiglossis sinuata

Somatic Hybridization by Fusion of Protoplasts : II. The combinations of Nicotiana tababum and N. glutinosa and of N. tabacum and N. alata

Studies on the Growth of Tobacco Roots : IV. Effect of oxygen concentration of media on the translocation of 14C assimilated into root

Studies on the Growth of Tobacco Roots : VII. Studies on the nicotine synthesis during the elongation of excised roots