PROTOPLAST FUSION

-

 

NAMA : ARYA WIRA WARDHANA

NPM : 21025010035

KELAS : A025

MATA KULIAH : BIOTEKNOLOGI

 

Protoplast Fusion


Enzymatic isolation is advantageous because protoplasts are gained at a high quantity, cells are not damaged and the osmotic conditions may be influenced. Enzymatic isolation may be carried out in two different procedures: two-step or one-step procedure. In the first step of the two-step procedure, individual cells are released with the help of commercial enzymatic preparations (e.g. macerozyme, macerase). The cells are released by degradation of the middle lamellas and the decay of tissue to individual cells. The free cells are then processed in the second step with the help of cellulases (cellulase Onozuka R-10, cellulysin) to protoplasts by dissolving the cell wall. The cells are exposed to the enzymes for a shorter time than at one-step isolation. One-step isolation is used more often, during which the mechanically loosened tissue (e.g. by cutting strips) is put into a mixture of enzymes (pectinase and cellulase, commercial preparations). For each plant object the optimal composition of enzymatic mixture is necessary as well as the optimal pressure of extraction media.

FACTORS INFLUENCING THE ISOLATION OF PROTOPLASTS

A successful isolation of protoplasts depends on many factors such as the source of tissue (leaves, cell suspension), plant species and cultivar, physiological condition of the donor plant, the composition of the enzymatic mixture and the period of enzymatic action, the osmotic characteristics of the extraction mixture and the individual steps during the isolation of protoplasts.

PROTOPLAST FUSION

Protoplast fusion leads to the formation of mixtures of genetic information – transfer of nuclear and cytoplasmic genetic information between plant species, genera, which could not be achieved during sexual crosses. It offers overcoming sexual barriers e.g. in breeding of agricultural plants. The aim of protoplast fusion is the transfer of genes controlling certain features e.g. from a wild growing plant into important agricultural crops. The characteristic aim of experiments with protoplast fusion has been to transfer: – genes of resistance to different virus and fungal diseases from wildly growing species, – cytoplasmic male sterility (CMS), – resistance to stress including tolerance to salinity, cold, drought, – resistance to insect parasites (synthesis of phytoalexins), – genes for synthesis of reserve proteins, vitamins, secondary metabolites of pharmaceutical importance. Using somatic hybrids created by protoplast fusion in plant breeding depends on the possibilities of selection and evidence of hybrids: morphological studies, cytological studies based on the number of chromosomes, using probes with DNA sequence when it is possible to find the size of the parent genome in hybrids, RFLP analysis and DNA-DNA hybridization, isoenzymes, chloroplast or mitochondrial DNA. The protoplast fusion must be induced immediately after the isolation of protoplasts prior to synthesis of a new cell wall. Undamaged protoplasts, practically immediately after washing out the used enzymes from the protoplast sample, regenerate a new cell wall and within a few days the cell division is renewed. The result of the fusion is a mixture of heterokaryon, homokaryon and unfused parent protoplasts. Somatic hybridization of plants includes four separate stages: – isolation of protoplasts (explant, enzymes, the period of enzymatic action, isolation method), – protoplast fusion (fusogen, viability and density of protoplasts), – selection and regeneration of plants, – analysis of regenerated plants. The physiological and genetic differences of partner cells determine the ability of the hybrid cells to survive. The elimination of a part of the genome of one or both partners during the first mitotic divisions is the way in which the hybrid cell prevents the negative effects of unsuitable genetic combination. Certain chromosomes, plastids or mitochondria may be eliminated. The method of somatic hybridization was used for the creation of genotypes of intergeneric character of “pomato” that do not exist in nature (MELCHERS et al. 1978), ArabidobrassicaDuring the protoplast fusion of distant taxa, non-viable products of fusion often appear or products which are not able to regenerate whole plants. Protoplasts isolated from juvenile embryos, young leaves and quickly growing calli fuse better

 

 












Sources :

Ahmed, M. A., Miao, M., Pratsinakis, E. D., Zhang, H., Wang, W., Yuan, Y., ... & Wu, B. (2021). Protoplast Isolation, Fusion, Culture and Transformation in the Woody Plant Jasminum spp. Agriculture, 11(8), 699.

Komentar

Posting Komentar

Postingan populer dari blog ini

In Vitro Propagation

-
 Arya Wira Wardhana / 21025010035 In Vitro Propagation of Date Palms (Phoenix dactylifera L.) The high consumption of date fruit has led to the cultivation of tropical dates in Indonesia since the last decade (Rahmadani et al., 2017). Date entrepreneurs hope that Indonesia can become a tropical date fruit producing country in the future, in addition to Thailand which has become a pioneer of tropical date cultivation in Southeast Asia (Apriyanti et al., 2016). The tropical dates cultivated in Thailand are KL-1 breeding results from the parent cultivars Degleet Nour and Barhee conducted by researchers at Maejo University (Apriyanti et al., 2016), while in Indonesia, date farms such as Jonggol Farm Bogor use tissue culture seeds originating from the UK. Until now, there is no laboratory in Indonesia capable of providing commercial-scale date palm tissue culture seeds. Tissue culture seeds are clonal or the same as the parent plant. Tissue culture seeds are produced through in vitro propag
Baca selengkapnya

ORGANOGENESIS IN PLANTS

-
 Arya Wira Wardhana / 21025010035 ORGANOGENESIS IN PLANTS Organogenesis is the process of forming cells, tissues or callus into buds and complete plants. Organogenesis is triggered by the presence of several components such as medium, endogenous components during explants in culture. Organogenesis can be grown from seeds, leaves or other plant parts so that it can develop into a perfect plant. Media is a determining factor in callus growth in tissue culture. Media contains nutrients that are usually needed by growing cells and are widely used, especially in the field of tissue culture. Media introduced by E. Vacin and F. Went since 1949 contains a number of inorganic nutrients, carbon sources, vitamins, growth regulators and organic supplements to meet the physiological needs of plant cells in tissue culture. Thus, in vitro propagation (micropropagation) is an alternative method that can be applied to obtain pathogen-free plants and allows it to be carried out on a large scale in a rel
Baca selengkapnya