Protoplast production- concepts and applications

Protoplast production (प्रोटोप्लास्ट उत्पादन):-

Protoplast Isolation (प्रोटोप्लास्ट पृथक्करण):- It has 2 main steps -

(इसके 2 मुख्य चरण हैं –)

a. Sterilization of Leaf (पर्ण का निर्जमीकरण)

b. Enzyme Treatment (एन्जाइम उपचार)

a. Sterilization of Leaf (पर्ण का निर्जमीकरण):- Soak the leaf in 70% ethanol for 1 minute. After this, keep this leaf in 2% NaOCl solution for 20 to 30 minutes. Now wash this leaf with distilled water for 3 minutes.

(पर्ण को 1 मिनट के लिए 70% एथेनॉल में डुबोकर रखते हैं। इसके पश्चात इस पर्ण को 20 से 30 मिनट के लिए 2% NaOCl विलयन में डुबोकर रखते हैं। अब इस पर्ण को 3 मिनट तक आसुत जल से धो लेते हैं।)

b. Enzyme Treatment (एन्जाइम उपचार):- Now this sterilized leaf is treated with 2 enzymes, pectinase and cellulase, respectively. The pectinase enzyme disintegrates the middle lamella. Cellulase enzymes decompose the cell wall. As a result protoplasts are obtained. To increase osmotic concentration, add 500 - 800 ml / L of sorbitol or mannitol.

(अब इस निर्जमित पर्ण को क्रमश: 2 एंजाइमों पेक्टीनेज व सेलुलेज से उपचारित कराया जाता है। पेक्टीनेज एन्जाइम मध्य पटलिका को विघटित कर देता है। सेलुलेज एन्जाइम कोशिका भित्ति को विघटित कर देता है। इसके फलस्वरूप प्रोटोप्लास्ट प्राप्त होते हैं। परासरणी सान्द्रता को बढ़ाने के लिए 500 – 800 ml/L सोर्बिटोल या मैनीटोल मिला देते हैं।)

Applications of protoplast production:- 

1. Study of Osmotic Behaviour:- 

Influence of different environmental factors on the osmotic behaviour can be studied using plant protoplasts.

2. Study of IAA Action:- 

> When growth promoters like IAA are ap­plied to plants, they act directly on plasma mem­brane of the cell and increase the permeability of the membrane to water resulting in cell elon­gation. This can be established by the use of protoplast in vitro.

> When IAA is applied to the plasmolyticum containing protoplasts they ex­pand rapidly and finally burst due to too much vacuolation. Further, it can be verified by using anti-auxins that sup­press this bursting, indicating that the site of action of IAA is the plasma-lemma of the plant cell.

3. Study of Plasma-lemma:-

When newly released protoplasts are placed in hypotonic solution or plain water, the proto­plasts burst within a second or if the protoplasts are dropped from a certain height on a glass slide, the same result will happen. So by this process, plasma-membrane can be isolated very easily from protoplast and a number of study on plasma-membrane can be investigated.

4. Study of Cell Wall Formation:-

The early deposition of cellulosic micro-fib­ril and their orientation at the protoplast surface can be followed using both light and electron mi­croscope and has also provided much basic infor­mation concerning cell wall biology.

5. Organelle Isolation:-

Protoplasts are very convenient material for the isolation of chloroplasts, mitochondria, nu­clei and even chromosomes. It has been demon­strated that chloroplasts particularly isolated from cereal protoplast have higher capacity for CO2 fixation than those obtained by mechanical grinding.

6. Study of Morphogenesis:-

> Isolated protoplast provides an ideal single cell system. Under suitable condition, protoplast regenerates its own wall and become the walled cells. Cell division followed by plant regeneration may occur from such unique single cell system either through organogenesis or embryogenesis.

> Plant regeneration is very important as well as significant for fusion experiment and for the experiment of genetic modification in proto­plasts.

7. Virus Uptake and Replication:-

> The plant virus interrelationships in the past were not clearly known due to lack of suit­able experimental systems that can easily infect the cells. But after the innovation of protoplast isolation and its culture, this problem is almost solved. 

> Protoplast can directly be inoculated with pathogenic virus in the medium. The pro­cess of uptake of virus particle, their replication inside the protoplasts and their mode of action at the molecular and cellular level are made pos­sible by the aid of protoplasts.

8. Study of Photosynthesis from Isolated Protoplast:- Elegant experiments to investigate various biophysical and biochemical aspects of photosynthesis in C3 and C4 plants have been carried out by a number of workers using protoplasts.

9. Isolation of Bacteriods from Root Nodule Protoplast:-

Viable bacteriods from root nodules of legu­mes has been isolated by first preparing nodule protoplast and then rupturing them either me­chanically or by lowering suddenly the concen­tration of the plasmolyticum in the surround­ing medium. This method ensures the freedom of the preparation of bacteria from the infection thread.

10. Induction of Mutation and Genetic Variability:-

> It has been repeatedly observed that plant cell in culture show a wide range of genetic diver­sity. This phenomena can be exploited by plant breeders and geneticists for inducing variability in protoplast culture. The recessive characters can be detected in the regenerated plants de­rived from haploid protoplasts. Therefore, hap­loid protoplast would make an ideal system for studying the effect of irradiation and for the in­duction of mutation by plating them in media supplemented with various chemical mutagens.

> From this method, mutant line can be se­lected.

11. Microorganism Transplantation:-

> Incorporation of microorganisms like bac­teria, blue-green algae, yeast etc. into proto­plasts has been attempted with the immediate objective of establishing endosymbiotic associa­tion with higher plant cells which may eventu­ally yield a plant having some beneficial activ­ity. Bacterial cell uptake by plant protoplasts has been investigated with species of Rhizobium and Spirillum.

> There are reports based on ultra- structural examinations that bacteria enter the cells by endocytosis and may become embedded in vesicles in the cytoplasm of protoplasts. Sim­ilar uptake studies were performed with yeast and blue green algae cells.

> Introduction of Anabaena variables and nitrogen fixing blue green al­gae Gleocapsa sp. into protoplasts has also been 15ported. However, nothing is known about the fate of the introduced microorganism, because there has been no reported evidence of survival or development of any organisms within the pro­toplasts.

12. Implantation of Chloroplast:-

> Plant protoplasts have ability to uptake the isolated chloroplasts by the process of endocy­tosis. Several reports have described uptake of chloroplasts. Chloroplasts isolated from Vaucheria dichotoma were implanted into carrot cell culture protoplasts. The chloroplasts may enter the cytoplasm enclosed in membrane-bound vesi­cles, although the enclosing membrane in some cases is absent.

> Biological evidence of chloroplast gene expression was presented but the ex­periments have not been confirmed. The inabil­ity and ability of chloroplasts to survive and mul­tiply in recipient protoplasts have not been un­equivocally demonstrated, although limited re­plication has been reported.

> Potentially the chloroplast uptake procedure offers an excellent approach to study chloroplast/ cytoplasm and nuclear interrelationships, genetics and physio­logical autonomy and specificity of functions of the organelles.

13. Transplantation of Nuclei:-

Isolated nuclei can be introduced into the protoplasts. Both intra and inter-specific nu­clear transplantation have been observed in Petu­nia hybrida, Nicotiana tabacum and Zea mays. Retention, normal function or degradation of the incorporated nuclei is not known. But it is really opening up new avenues for the study of nuclear- cytoplasmic interaction if fertile plants with for­eign nuclei could be regenerated from such pro­toplasts.

14. Transplantation of Chromosome:-

The uptake of isolated metaphase chromo­somes has proven successful in plant protoplast. This procedure provides a valuable method for genetic information transfer and gene analysis.

15. Somatic Hybridization:-

The main objective of protoplast culture lies in the possibilities of fusion of one protoplast with another. Normally isolated protoplasts do not fuse with each other, but in presence of fu­sion inducing agent like PEG, a protoplast of one species can be fused with one of a differ­ent species, thus a hybrid protoplast will be pro­duced.