Somatic Hybridization and Cybridization
Sexual hybridization in higher plant is a valuable tool for the conventional plant breeding to improve cultivated crops. However, many desirable combinations of characters can not be transmitted through conventional methods of genetic manipulation. Secondly, conventional hybridization is limited to only very closely related species and was total failure for distantly related species as well as in sexually incompatible species. However, by using a protoplast fusion technology , it possible to fuse two genotypically different by protoplast to obtain para sexual hybrid protoplast.
Definition of Somatic Hybridization:
It is fusion between isolated somatic protoplasts under in vitro conditions and subsequent development of their product to a hybrid plant is known as somatic hybridization.
Plasmid and mitochondrial genomes are inherited maternally in sexual crossings. Through the fusion process the nucleus and cytoplasm of both parents are mixed in the hybrid cell. This results in various nucleo- cytoplasmic combination. Sometimes interaction in the plastome and genome contribute to the formation of cybrid. Cybrids in contrast to conventional hybrids, possesses a nucleus genome from only one parent but cytoplamsmic gene
The process of protoplast fusion resulting in the development of cybrid is called as Cybridization.
In Cybridization heterozygosity of extra-chromosomal material can be obtained, which has direct application in plant breeding. Studies during last decade have revealed that the process of protoplast fusion may be a useful too for the induction of genetic variability and combination of traits which do not exist in nature.
Isolation of Protoplast:
Methods of Protoplasts isolation can be classified into three main groups.
a) Mechanical :
Mechanical isolation is done by cutting plasmolysed tissue with a sharp edged knife and releasing the protoplasts by deplasmolysis .The protoplasts isolated are few in number. Generally, protoplasts were isolated from highly vacuolated cells of storage tissues( Onion, bulbs, scales, radish root, mesocarp of cucumber and beet root) .
b) Sequential Enzymatic (two step):
Takebe et al. (1968) employed sequential or two step procedure for isolating mesophyll protoplasts using commercial preparation of enzymes. The sequential approach involves initial incubation of macerated plant tissues with pectinase which, in turn, are then converted into protoplasts by cellulose treatment.
c) Mixed Enzymatic Procedure:
Cocking (1968) mixed two enzymes together and isolated protoplasts in one –step. In this mixed enzymatic approach plant tissues are plasmolysed in the presence of a mixture of pectinases and cellulases, thus inducing concomitant separation of cells and degradation of their walls to release the protoplasts directly.
Source of Protoplasts:
The leaf is the most convenient and popular source of plant protoplast because it allows isolation of a large number of relatively uniform cells. Protoplasts isolation from leaves involve five basic stages: a) Sterilization of leaves, b) removal of epidermal cell layer c) Pre-enzyme treatment d) incubation in enzyme and e) isolation by filtration and centrifugation.
In case of monocots, leaf material is cut into small places (1mm2) and then combined with vacuum infiltration. This procedure allows adequate infiltration of enzymes into leaf cells. As soon as the vacuum is removed the leaf piece will sink and eventually release the mesophyll protoplasts.
ii) Callus Culture:
Young actively growing callus is subcultured and used after two weeks for protoplasts isolation.
iii) Cell Suspension Culture:
A high –density cell suspension is centrifuged. After removing the supernant, cell are incubated in enzyme mixture ( cellulose + pectinase) in a culture flask placed on a platform shaker for 6 hrs to overnight depending on to the concentration of enzymes. A lower concentration of enzymes helps to prevent the formation of aggregates in the cell suspenion in order to obtain better yield.
iv) Preconditioned Plant Materials:
Mesophyll protoplast of some crop plants have a low morphogenetic response. This is because of the fact that the physiological state of growth of a donor plant under natural condition largely affect the regeneration potential of protoplasts in this system. on the contrary, tissue cultured regenerated plants are maintained under uniform physiological conditions and therefore provides materials preconditioned for protoplasts isolaton, and regeneration. This approach is particularly essential for regeneration of potato protoplasts.
Test for Viability of Protoplast:
Cell wall formation, cell division, callus formation, etc. depends upon the viability of protoplast. The most frequently used staining methods for assessing protoplast viability are fluorescens diactate ( FDA), phenosafranine. FDA dissolved in 5.0 mg/ml acetone is added to the protoplast culture at 0.01% final concentration. The chlorophyll from broken protoplasts fluoresces red. Therefore, the percentage of viable protoplast in a preparation can be easily calculated. Phenosafranin, also used at final concentration of 0.01% is specific for dead protoplast. As soon as the strain is mixed with protoplast preparation, the invibale protoplasts strain red and viable protopalsts remain unstained.
Formation of Cell Wall:
The process of cell-wall formation may be completed in two to several days although protoplast in culture generally starts regenerating a cell wall within a few hours after isolation. The regeneration of cell wall can be detected by using Calcalfluor White (CFW) florescence strain. The freshly formed cell wall is composed of loosely arranged microfibrils , the process requiring an exogeneous supply of a readily metabolised carbon source in the nutrient medium.
Development of Callus or Whole Plant:
Soon after the formation of wall around the protoplasts, the reconstituted cells show considerable increase in size and first division generally occurs within a week. Subsequent division give rise to cell colonies. After 2-3 weeks macroscopic colonies are formed which can be transferred to an osmotic free medium to develop a callus. The callus may be induced to undergo organogenic differentiation or whole plant regeneration following a appropriate procedure.