PRODUCTION OF NEW CULTIVARS

PRODUCTION OF NEW CULTIVARS

Consider about the production of new cultivars there are three major parts,

• Somatic Hybridization
• Protoplast Fusion
• Somaclonal Variation

Somatic Hybridization

                  Production of hybrid plants through the fusion of protoplasts of two different plant species/varieties is called somatic hybridization, and such hybrids are known as somatic hybrids.

The technique of somatic hybridization involves the following four steps:

(1) Isolation of protoplasts.

(2) Fusion of the protoplasts of desired species/varieties.

(3) Selection of somatic hybrid cells.

(4) Culture of the hybrid cells and regeneration of hybrid plants from them.

(1) Isolation of protoplasts

                    Isolation of protoplasts is readily achieved by treating cells/tissues with a suitable mixture of cell wall degrading enzymes. Usually, a mixture of pectinase or macerozyme  and cellulase  is appropriate for most plant materials.

                    Hemicellulase may be necessary for some tissues. Generally, crude commercial preparations of enzymes are used. The pH of enzyme solution is adjusted between 4.7 and 6.0 and the temperature is kept at 25-30°C.

                    Usually, 50-­100 m mol 1-1 CaCl₂ is added to the osmoticum as it improves plasma membrane stability. The cells and tissues are incubated in the enzyme mixture for few to several (generally, 16-18) hours; naked protoplasts devoid of cell wall are gradually released in the enzyme mixture.

                    Protoplasts have been isolated from virtually all plant parts, but leaf mesophyll is the most preferred tissue, for this purpose. In general, fully expanded leaves are surface sterilized,

 their lower epidermis is peeled off with a pair of forceps and the peeled areas are cut into small pieces with a scalpel and suspended in the enzyme mixture. When epidermis cannot be peeled, leaf may be cut into pieces and treated with the enzyme mixture; vacuum infiltration may be used to facilitate the entry of enzymes into the tissues.

                    After the period of incubation, protoplasts are washed with a suitable washing medium in order to remove the enzymes and the debris. The protoplasts may be cultured on a suitable medium in a variety of ways:

·         Bergmann's plating technique (in agar medium)

·         In a thin layer of liquid medium.

·         In small microdrops of 50-100 µl.

                     Protoplasts readily regenerate cell wall (within 2-4 days) and undergo mitosis to form macroscopic colonies, which can be induced to regenerate whole plants. The conditions for isolation and culture of protoplasts and regeneration of complete plants have been standardized for a large number of plant species, but cereals still present some problems.

                   Generally, MS and B5 media, and their modifications are used for protoplast culture. The media are supplemented with a suitable osmoticum and, almost always, with an auxin and a cytokinin, their types and concentrations depending mainly on the plant species. After 7-10 days of culture, protoplasts regenerate cell wall and the osmolarity of medium is gradually reduced to that of normal medium.

The macroscopic colonies are transferred onto normal tissue culture media. Protoplasts are very sensitive to light; therefore, they are cultured in diffuse light or dark for the first 4-7 days.

(2) Protoplast Fusion

                    A number of strategies have been used to induce fusion between protoplasts of different strains/species; of these the following three have been relatively more successful. Protoplasts of desired strains/species are mixed in almost equal proportion; generally they are mixed while still suspended in the enzyme mixture.

                    The protoplast mixture is then subjected to a high pH and high Ca²⁺ concentration at 37°C for about 30 min (high pH­ high Ca²⁺ treatment). This technique is quite suitable for some species, while for some others it may be toxic.

                    Polyethylene glycol (PEG) induced protoplast fusion is the most commonly used as it induces reproducible high frequency fusion accompanied with low toxicity to most cell types. The protoplast mixture is treated with 28-50% PEG for 15-30 min, followed by gradual washing of the protoplasts to remove PEG; protoplast fusion occurs during the washing.

                    The washing medium may be alkaline (pH 9-10) and contain a high Ca²⁺ ion concentration this approach is a combination of PEG and high pH-high Ca²⁺ treatments, and is usually more effective than either treatment alone. PEG is negatively charged and may bind to cations like Ca^2+, which, in turn, may bind to the negatively charged molecules present in plasma lemma; they can also bind to cationic molecules of plasma membrane.

                    During the washing process, PEG molecules may pull out the plasma lemma components bound to them. This would disturb plasma lemma organisation and may lead to the fusion of protoplasts located close to each other.

                    The above fusion techniques are nonselective in that they induce fusion between any two or more protoplasts. A more selective and less drastic approach is the electrofusion technique, which utilizes low voltage electric current pulses to align the protoplasts in a    single row like a pearl-chain. The aligned protoplasts can be moved, with a micromanipulator, and pairs of protoplasts may be isolated in individual microelectrofusion chambers.

                    The pairs of protoplasts can be fused by a very brief (few microseconds) pulse of high voltage. Alternatively, the protoplasts may be subjected to mass electrofusion; in such a case the population of protoplasts is subjected to high voltage after they are brought close to each other by the low voltage current.

                    The high voltage creates transient disturbances in the organisation of plasma lemma, which leads to the fusion of neighbouring protoplasts. The entire operation is carried out manually in specially designed equipment, called electroporator. Many workers feel that this fusion technique is more desirable than the others for a number of important reasons.

(3) Selection of Hybrid Cells

                    The protoplast suspension recovered after a treatment with a fusion inducing agent (fusogen) consists of the following cell types:

·         unfused protoplasts of the two species/strains,

·         products of fusion between two or more protoplasts of the same species (homokaryons),

·         'hybrid' protoplasts produced by fusion between one (or more) protoplast(s) of each of the two species (heterokaryotis).

                   

                    In somatic hybridization experiments, only the heterokaryotis or hybrid protoplasts, particularly those resulting from fusion between one protoplast of each of the two species, are of interest. However, they form only a small proportion of the population (usually 0.5-10%).

                     Therefore, an effective Strategy has to be employed for their identification and isolation. This step is called the selection of hybrid cells, and is the most critical, and is still an active area of investigation.

                      A number of strategies have been used for the selection of hybrid protoplasts.

(i)  Some visual markers, e.g., pigmentation, of the parental protoplasts may be used for the identification of hybrid cells under a microscope; these are then mechanically isolated and cultured. For example, the protoplasts of one species may be green and vacuolated (from mesophyll cells), while those of the other may be nonvacuolated and nongreen (from cell cultures). Where such features are not available, the protoplasts of two parental species may be separately labelled with different fluorescent agents.
This approach is time consuming, and requires considerable skill and effort. Several workers have attempted to devise systems, which specifically select for hybrid cells.

(ii) These systems, exploit some properties (usually, deficiencies) of the parental species, which are not expressed in the hybrid cells due to complementarily between their genetic systems. These properties may be sensitivity to culture medium constituents, antimetabolites, temperature, etc. inability to produce an essential biochemical (auxotrophic mutants), etc. These properties may be naturally present in the parental species or may be artificially induced through mutagenesis/genetic engineering.

                    For example, protoplasts of Petunia hybrida form calli on the MS medium, while those of P. parodii produce only small cell colonies. Further, inhibits cell division of P. hybrida protoplasts but it has no effect on those of P. parodii.

                   Thus protoplasts of both these Petunia species fail to produce macroscopic colonies (calli) on MS medium supplemented with µg ml-l actinomycin D. However their hybrid cells (P. hybrida + P. parodii; somatic hybrids are denoted by a + sign divide normally on this medium to produce macroscopic colonies. This selection strategy exploits those natural properties of the two parental species, which show complementation in the hybrid cells.

                    These strategies are simple, highly effective and the least demanding. Recently, genetic engineering has been used to transfer resistance to an antibiotic/herbicide in one fusion parent and that to another one into the other parent; the hybrid cells are selected using a medium containing both the concerned antibiotics/herbicides.

A more general and widely applicable strategy is,

(iii) To culture the entire protoplast population without applying any selection for the hybrid cells. All the types of protoplasts form calli; the hybrid calli are later identified on the basis of callus morphology, chromosome constitution, protein and enzyme banding patterns, etc.

(4) Regeneration of Hybrid Plants

                   Once hybrid calli are obtained, plants are induced to regenerate from them since this is a: prerequisite for their exploitation in crop improvement.

                    Further, the hybrid plants must be at least partially fertile, in addition to having some useful property, to be of any use in breeding schemes. The culture techniques have been refined to a state where plant regeneration has been obtained in a number of somatic hybrids.

Somaclonal Variation

                                   

                     Shoot tips or axillary buds are used for direct propagation on culture medium without intervening callus phase. It has been shown in recent years, that regeneration from callus, leaf explants or plant protoplasts leads to the generation of considerable variation, described as ‘somaclonal variation’. This variation includes aneuploids, sterile plants and morphological variants, sometimes involving traits of economic importance in case of crop plants. This variation received increased attention and excitement during 1980s in view of its potential in crop improvement programmes. The usefulness of this variability in crop improvement programmes, was first demonstrated through the recovery of disease resistant plants in potato (resistance against late blight and early blight) and sugarcane.

                    In plants regenerated from callus, not only variability involving both nuclear and organellar DNA is observed, but variability even in chromosome number is observed in long-time cell cultures. This is exhibited in callus and in plants regenerated from the callus.

                    It may be necessary to remember that the above variation may be transient (epigenetic) or genetic; only the latter is transmitted to the next generation and is thus important for crop improvement of a sexually propagated crop. In several crops R₀, R₁ and R₂ progenies were subjected to genetic analyses and 3:1 segregation was observed, leading to the isolation of true breeding variants.

                    Although the details of the genetic basis of somaclonal variation in most crops are still largely unknown, variation in structure and number of chromosomes has been suggested to be one possible basis. Polyploidy, aneuploidy, translocations, inversions and deletions have been reported in several cases. Meiotic crossing over involving symmetric and asymmetric recombination could also be responsible for a part of the variation observed in the regenerated plants. Methylation at the DNA level in tissue culture has also been considered as a possible reason for ‘somaclonal variation’.

                    Somaclonal variation has actually been used in plant breeding programmes. A number of plant species, where useful somaclonal variation.

Mechanism of Somaclonal Variations

1. Genetic (Heritable Variations)

              Genetic varations occucrs due to

•         point mutations (e.g., Adh mutants in wheat)

•         cytoplasmic (maternal inheritance)

•         gene amplification (e.g., incr. gene copy no.)

•         activation of transposable element

•         cytogenetic (changes to genome structure)

                                        aneuploidy – gain or loss of 1 or more chromosomes

                                        polyploidy – gain or loss of an entire genome

                                        translocation – arms of chromosomes switched

                                        inversion – piece of chromosome inverted

2.  Epigenetic (Non-heritable Variations)

Variations generated during tissue culture

Caused by temporary phenotypic changes

Usually undesirable in a breeding program, not always undesirable in propagation.

Steps involved in induction and selection of Somaclonal Variations

                                 Callus Tissue

     Organogenesis                                Somaclonal Variants

     Regenerated plants                         Hardening and Selfing

                    

Physiological Causes, Genetic Causes and  Biochemical Causes  affect to  Somaclonal Variations

Ø  Physiological Cause

Exposure of culture to plant growth regulators.

Culture conditions

Ø  Genetic Cause

1.      Change in chromosome number

§  Euploidy: Changes chromosome Sets

§  Aneuploidy: Changes in parts of chromosome Sets

§  Polyploidy: Organisms with more than two chromosome sets

§  Monoploidy: Organism with one chromasomes set

2.      Change in chromosome structure

§  Deletion

§  Inversion

§  Duplication

§  Translocation

3.      Gene Mutation

§  Tansition

§  Transversion

§  Insertion

§  Deletion

      4.  Plasmagene Mutation

      5.  Transposable element activation

      6.  DNA sequence

o   Change in DNA

§  Detection of altered fragment size by using Restriction enzyme

o   Change in Protein

§  Loss or gain in protein band

§  Alteration in level of specific protein

o   Methylation of DNA

§  Methylation inactivates transcription process.

 

 Ø  Biochemical Cause

•         Lack of photosynthetic ability due to  alteration in carbon metabolism

•         Biosynthesis of starch via carotenoid pathway

•         Nitrogen metabolism

•         Antibiotic resistance.

Detection and Isolation of Somaclonal Variants

                         Identification  of possible somaclonal variants at anearly stage of development is considered to be very useful for quality control in plant tissue culture, transgenic plant production and in the introduction of variants.

1.      Analysis of morphological characters

•         Qualitative characters: Plant height, maturity date, flowering date and leaf size

•         Quantitative characters: yield of flower, seeds and wax contents in different plant parts

2.      Variant detection by cytological Studies

•         Staining of meristematic tissues like root tip, leaf tip with feulgen and acetocarmine provide the number and morphology of chromosomes.

3.      Variant detection by DNA contents

•         Cytophotometer detection of feulgen stained nuclei can be used to measure the DNA contents

      4. Variant detection by gel electrophoresis

           Change in concentration of enzymes, proteins and hemical products like pigments,              amino acids  and alkaloid can be   detected by their  electrophoretic   pattern

      5.  Detection of disease resistance variant

               Pathogen or toxin responsible for disease resistance can be used as selection agent 

          during culture.

      6.  Detection of  herbicide resistance variant

            Plantlets generated by the addition of herbicide to the cell culture system can be used as  herbicide resistance plant.

      7.  Detection of  environmental stress tolerant variant

            Selection of high salt tolerant cell lines in tobacco

            Selection of water-logging and drought resistance  cell lines in tomato

            Selection of temperature stress tolerant in cell lines in pear.

            Selection of mineral toxicities tolerant in sorghum plant (mainly for aluminium toxicity)