List of top two experiments on plant tissue culture:- 1. Isolation of Explants, Establishment and Maintenance of Callus 2. Culture of Anthers and the Establishment of Haploid Plants.
Experiment # 1. Isolation of Explants, Establishment and Maintenance of Callus:
Explants isolated from the tissues of higher plants are brought into culture. It requires a nutrient medium consisting of a mineral salts mixture, a carbon source and vitamins.
In addition, phytohormones or their synthetic counterparts are required to initiate and maintain cell division, occasionally other organic supplements — for instance amino acids or hexitols — are necessary to ensure the prolonged growth of the excised tissue to give an established callus.
On suitable media, tissue fragments from most of the plant parts will start to proliferate rapidly and, finally, produces undifferentiated cell mass i.e. callus.
Materials and Equipments:
1. Preparation of media:
In general, each medium contain five distinct categories of ingredients viz. macro-elements, micro-elements, chelated iron, phytohormones (auxin and cytokine) and plant organics. Usually the stock solution way prepared earlier and stored in refrigerator. When required, these stock solutions were mixed together in proportions for preparation of an artificial culture medium (Table 1).
To make up the culture medium, 100 ml of the AB mixture is added to 500 ml of distilled water, then 10 ml of soln. C and 10 ml of solution D, 1 ml of solution E and 30 g of sucrose are added to the said mixture.
Finally, the volume of the medium is made up to 1,000 ml by adding distilled water. The pH of the medium is adjusted to 5.8 (with N/10 KOH and N/10 HCI). To prepare solid medium, 20 gm., of powdered agar is added and the mixture was autoclaved for sterilization (15 minutes at 15 lbs. pressure).
Usually, Solution D and Solution E were added to the medium after autoclaving. The Solution D (i.e. phytohormones) and Solution E (i.e. plant organics) were sterilized by passing through millipore filter.
2. A number of instruments and glass goods required are:
(i) Culture tubes,
(ii) Tissue paper,
(iii) Petridishes,
(iv) Aluminium foil,
(v) Forceps,
(vi) Scalpels,
(vii) Distilled water, etc.
All these materials were wrapped with paper and then sterilized in autoclave or in hot air oven (except distilled water).
3. Plant tissue explants (young bud, twig and roots etc.).
4. Incubation of isolated explants implanted on selected media at 25±1°C under illumination in incubation room.
Procedure:
The callus culture from excised carrot tap root is described here as a type case:
1. A fresh tap root of carrot is taken and thoroughly washed under running tap-water to remove all surface debris;
2. The root is then dipped into 5% Teepol for 5 to 10 minutes for surface sterilization and then the root is washed in sterilized distilled water;
3. The root is further surface sterilized by immersing in 70% ethanol (v/v) for 40-60 seconds, followed by 5% sodium hypo-chloride (v/v) for 20-30 minutes;
4. The root is finally washed in sterilized distilled water (three times);
5. Then with the help of sterilized scalpel, a thin section of root is made aseptically on a sterilized petridish, which was subsequently immersed in 5% sodium hypochlorite solution for 5-10 minutes and finally washed by sterilized distilled water and placed in a series on a sterilized petridish;
6. Then carefully cambial tissue explant was removed aseptically from the slices and then quickly transfer the same into medium, for callus growth;
7. The process of inoculation of medium by sterilized tissue explants and subsequent incubation is very much identical to the microbial isolation and incubation technique.
8. At least after 4-6 weeks, callus growth can be notices on the edge of tissue explant thus implanted on desired medium;
9. Then callus tissue was removed from the culture tube and segmented aseptically for subsequent subculture of callus. Prolong culture of callus will produce large mass of callus which is either used for regeneration organogenesis or commercial extraction of secondary metabolites.
Detailed stages of callus culture procedure from carrot root is given in Fig. 1.1.
Experiment # 2. Culture of Anthers and the Establishment of Haploid Plants:
Anthers may be removed aseptically from sterilized flower buds and placed in culture. A proportion of pollen within these anthers — particularly those from Solanaceous species — grow and proceed through a series of developmental stages, eventually giving rise to haploid embryos.
This phenomenon has been designated as “androgenesis”. In addition, there is also possibilities of haploid embryo formation, the ultimate resultant is the formation of regenerated haploid plant.
Materials and Methods:
1. Flower buds (freshly collected) of Nicotiana tabacum;
2. Modified Msculture media;
3. Sterilized glass goods like petridish, beakers, glass vial with lids;
4. Sterilized scalpel, forceps, Bunsen burner etc.;
5. Surface stimulants like 5% teepol, 70% ethanol (v/v), sterilized distilled water.
Procedure:
The steps of anther culture are:
1. The flower buds of Nicotiana tubacum (measuring 15-20 mm), is collected from flowering plants only;
2. Then selected buds are taken for surface sterilization by immersing in 5% teepol solution for 40-60 seconds. The buds are immediately washed in sterilized distilled water;
3. Subsequently, the buds are immersed in 70% ethanol (v/v) for 10 seconds followed by 2% sodium hypochlorite (v/v) for 10 minutes. Finally, the buds were washed thoroughly in sterilized distilled water (three times) and transferred to pre-sterilised petridishes;
4. By sterile forceps and scalpel, the anthers were aseptically removed and then either anthers are directly placed on culture media aseptically or they are crushed for isolation of pollen through centrifugation in sterilized buffer and then suspension of pollen is plated on agar media for embryogenesis;
5. The cultures are kept initially in dark. After 3-4 weeks they undergo embryogenesis or androgenesis and then the culture vials are kept in light for proliferation of callus tissues;
6. At this stage, the culture was incubated at 24-28°C for 14 hrs. in light at about 2,000 lux. intensity, then plantlets are regenerated from young differentiated embryo.
The detailed stages of anther culture techniques are shown in Fig 1.2. 