Induced Fusion

5.2. Induced Fusion

In actual practice, however, the somatic hybridization is invariably exploited for the fusion of protoplasts by following two distinct routes adopted quite often, namely : (a) interspecific fusion — using two different species ; and (b) employing two diverse sources within the same species. Therefore, to accomplish this cardinal objective one needs to adopt induced fusion having an appropriate agent (fusogen) ; whereas the spontaneous fusion may not prove to be effective and useful.

Examples :

(a) In animals : Sendai virus (inactivated) — is required to intiate the process of induced fusion, and

(b) In plants : PEG* treatment ; NaNO 3 (at high pH) + Ca 2+ (high concentration) ; and electri- cal impulse — are needed to achieve the phenomenon of induced fusion.

It has been duly observed that in the plant kingdom the respective inducing agent, as stated above, exert its action in two stages, namely :

(a) Bringing together the protoplasts, and (b) Causing them to adhere to one another for affording fusion ultimately.

Fig. 2.5 vividly illustrates the various means to accomplish successfully the fusion of plant protoplasts spread over to almost three decades (1970 to 2000).

NaNO (High pH) 3 +

Ca ( High conc.) 2+ Electrical Impulse

Polyethylene Membrane Modifying Glycol (PEG)

Substances

AB

Fig. 2.5. Various Experimental Parameters Employed for Induction of Protoplasts Fusion.

* PEG : Polyethylene glycol ;

GENETIC RECOMBINATION

The following four modes of treatment essentially associated with the production of induced fusion shall be treated individually as under :

(i) Sodium Nitrate (NaNO 3 ) Treatment

(ii) Calcium Ions (Ca 2+ ) Treatment at High pH, (iii) Polyethylene Glycol (PEG) Treatment, and (iv) Electrical Impulse (Fusion).

5.2.1. Sodium Nitrate (NaNO 3 ) Treatment

The NaNO 3 -treatment method was employed gainfully for the fusion of protoplasts obtained from the root tips of maize and coat seedlings ; however, the method is not preferred so abundantly on account of its rather low frequency of fusion, specifically when extremely vacuolated mesophyll protoplasts are employed. The various steps that are being adopted sequentially are illustrated in Fig. 2.6.

Step 1 : An ‘aggregation mixture’ comprising of sodium nitrate (5.5% w/v) in sucrose solution (10% w/v) is first prepared into which the isolated protoplasts are duly suspended.

The aforesaid mixture significally serves as a fusion-inducing mixture, and affords fusion upon adequate incubation in a water bath maintained at 35 ± 2°C. However, one may accomplish higher

frequency of fused protoplasts by subjecting the resulting mixture to centrifugation and the ‘pellet’

resuspended and reincubated for at least one or more additional cycles.

Step 2 : The fusion-inducing mixture obtained in step 1 is promptly replaced by a liquid-

culture medium ; and the protoplasts contained in this mixture are reincubated (35 ± 2°C). If so re- quired the aforesaid cycle may be repeated at least one or two times before plating the protoplasts on

a solid medium. Thus, the fusion of protoplasts may be monitored adequately at various stages via intensive examination under an inverted microscope.

PHARMACEUTICAL BIOTECHNOLOGY

Nicotiana longsdorfii

Nicotiana glauca

Leaf meosphyll cells treated with enzymes to digest away the cell walls

Protoplasts are mixed and suspended in NaNO to cause 3

cell fusion

Protoplast suspensions plated on agar medium with no. auxin or cytokinin ; only fused hybrid cells grow

Hybrid cells differentiate

to form shoots

Shoots grafted onto a

Seeds germinate to

parent plant ;

produce seedlings

similar to sexually to produce fertile flowers and seeds

the hybrid scion matures

produced amphidiplold

Fig. 2.6. Various steps Involved in Sodium Nitrate Treatment for Production of Interspecific Somatic Hybrids in the genus Nicotiana.

[Adopted from : Biotechnology and Genomics, PK Gupta (2004)].

GENETIC RECOMBINATION

5.2.2. Calcium Ions (Ca 2+ ) Treatment at High pH

Bhojwani and Razdan* (1983) devised a method involving centrifugation (spinning) of the protoplasts taken up in a fusion-inducing solution (0.05 M CaCl 2 . 2H 2 O in 0.4 M mannitol at pH 10.5) for 30 minutes at 50°C, after which the tubes were incubated at water-bath maintained at 37°C for a duration ranging between 40-50 minutes, which caused fusion of protoplasts to the extent of 20-50%. However, the method proved to be superior in comparison to other methods in certain cases, whereas the high pH (10.5) turned out to be too toxic in other instances.

5.2.3. Propylene Glycol (PEG) Treatment

PEG, as a fusogen, has been employed both gainfully and successfully in carrying out the protoplasts fusion in a variety of crops since 1974. Interestingly, this technique essentially attributes high frequency of fusion having reproducible results as well as relatively lower cytotoxity. Neverthe- less, this technique may be extended quite effectively and fruitfully for the fusion of protoplasts be- longing to rather unrelated/divergent plant taxanomy, such as : suyabean-barley ; soyabean-maize ; and soyabean-tobacco.

The agglutination** of protoplasts, in the course of PEG-treatment, may be accomplished by adopting either of the two following methods :

Method-1 : Protoplasts in Macroquantities

In a situation, when the protoplasts are available in macroquantities, 1 ml of the prepared cul- ture medium along with the two types of protoplasts is carefully added into 1 ml of 56% solution of PEG, and finally the contents of the tube is shaken thoroughly for 5 seconds only. Consequently, the protoplasts are allowed to undergo sedimentation for a span of 10 minutes, washed with the liquid growth (culture) medium once or twice, and finally examined for successful achievement of both agglu-

tination and fusion. Method-II : Protoplasts in Microquantities

In case, the protoplasts are available in microquantities, the technique of drop cultures may be employed effectively. The two types of protoplasts, in equal quantities [i.e., 100 µ L each or 4 to 6 drops] are mixed and placed carefully in a Petri-dish, and allowed to settle at room temperature (20 ± 2°C) for a duration of 5 to 10 minutes. Now 50 µ L PEG (2-3 drops) are carefully added from the

periphery in each Petri-dish, that are subjected to incubation for 30 minutes at room temperature (24°C), which ultimately leads to agglutination of the protoplasts. Subsequent to the PEG treatment the result-

ing protoplasts are washed meticulously during which the protoplasts fusion takes place largely. At this stage the PEG is replaced by the culture medium to pemit the ultimate growth of the fused protoplasts.

5.2.4. Electrical Impulse (Fusion)

It has been observed that when the protoplasts are duly placed into a small culture vessel having electrodes, and an adequate potential difference is applied, the ensuing protoplasts would get accumu- lated in between the electrodes. At this critical juncture if one applies an extremely short electrical impulse (shock), it will afford the induction of protoplasts fusion as depicted in Fig. 2.7 including the various stages involved from (a) through (d).

* Bhojwani SS and MK Razdan, Plant Tissue Culture : Theory and Practice, Elsevier, Amsterdam, 1983. ** Causing the cells to clump together.

PHARMACEUTICAL BIOTECHNOLOGY

undergoing fusion

() c () d

Fig. 2.7. Various Stages from (a) to (d) Depicting Protoplast Fusion Induced by Application of Electrical Impulse (Shock)