Basic knowledge of research

 

Types of Culture

Callus Culture:

Callus tissue means an unorganised prolifer­ative mass of cells produced from isolated plant cells, tissues or organs when grown aseptically on artificial nutrient medium in glass vials un­der controlled experimental conditions.

Principles of Callus Culture:

Aseptic preparation of plant material, Selection of suitable nutrient medium sup­plemented with appropriate ratio of auxins and cytokinins or only appropriate auxin, and Incubatif culture under controlled phy­sical condition.

Requirement:-Carrot, Petri plates, Flasks, Scalpel, Incubator, Teepol, ethanol, Sodium hypochlorite, D/W, MS media

 

Protocol of Callus Culture:

Callus tissue can be induced from different plant tissues of many plant species. Carrot is a highly standardized material.

(1)   A fresh tap root of carrot is taken and wash­ed thoroughly under running tap water to remove all surface detritus

(2)    The tap root is then dipped into 5% ‘Tee­pol’ for 10 minutes and then the root is washed.

(3)    The tap root is surface sterilized by immers­ing in 70% v/v ethanol for 60 seconds, fol­lowed by 20-25 minutes in sodium hypo­chlorite (0.8% available chlorine).

(4)    The tap root is surface sterilized by immers­ing in 70% v/v ethanol for 60 seconds, fol­lowed by 20-25 minutes in sodium hypo­chlorite (0.8% available chlorine).

(5)    The root is washed three times with ster­ile distilled water to remove completely the hypochlorite.

(6)  A series of transverse slice 1 mm in thickness is cut from the tap root using a sharp scalpel. Each piece is transferred to another ster­ile petridish. Each piece contains a whitish circular ring of cambium around the pith. An area of 4mm² across the cambium is cut from each piece.

(7)    The cotton plug from a culture tube is removed and flamed the uppermost 20 mm of the open end. While holding the tube at an angle of 45°, an explants is transferred using forceps onto the surface of the agarified nutrient medium. Nutrient medium is Gamborg’s B₅ or MS medium supplemented with 0.5 mg/L 2, 4-D.

(8)    Date, medium and name of the plant are written on the culture tube by a glass marking pen or pencil.

(9)Culture tubes after inoculation are taken to the culture room where they are placed in the racks. Cultures are incubated in dark at 25°C.Usually, after 4 weeks in culture the ex- plants incubated on medium with 2, 4-D will form a substantial callus.

(10)The whole callus mass is taken out aseptically on a sterile petridish and should be divided into two or three pieces. Each piece of callus tissue is transferred to a tube containing fresh same medium

 

 

 

Organ Culture

Any plant  organ can serve as an explants to initiate culture

Organ	Culture type
Shoot	Shoot tip culture
Root	Root tip culture
Leaf	Leaf culture
Flower	Anther/Overy/Ovule culture

 

Root culture :- The culture of excised radical tips of aseptical tips of aseptically germinated srrds in a liquid or solid medium where they are induced to grow independently under controlled condition.

Principle:- Intact in vivo plants are not suitable for the isolation of intact root tips because the roots of ‘the plant are buried deeply in the soil. Again, root tips from young seedlings are very sensitive to toxic sterilants. So it is better to avoid the surface sterilization of young root tips for the es­tablishment of root cultures.

Requirement:-Petri plates, Flasks, Incubator, White`s basal medium

Protocol:-

Initiation of Isolated Root Culture:

(1) Seeds are surfaced sterilized by the conven­tional methods and germinated on moist fil­ter paper or White’s basal medium at 25°C in the dark (Fig 2.1).

(2) When the seedling roots are 20 to 40 mm in length, 10 mm apical tips (tip inoculum) are excised with a scalpel and each transferred to 40 ml of liquid medium contained in 100 ml wide-necked Erlenmeyer flasks.

(3) Flasks are incubated at 25°C in the dark.

Initiation of Clones:

The root material derived from a single radi­cle tip could be multiplied and maintained in continuous culture.

The protocol is given below:

(1) Establish a root culture from a radical tip of a seed as described above.

(2) Transfer a 10-day-old established root cul­ture to a sterile petri dish containing ster­ile medium. Next, using flamed scissors, cut the main axis of root into a number of pieces (each piece is called sector inoculum or initial), each bearing four or five young laterals.

(3) Transfer the individual sector inoculum aseptically to a flask liquid medium and in­cubate in dark at 25°C.

(4) Such sector culture can be used to initiate further tip culture using 10mn apical tips of laterals of a growing sector inoculums or the growing sector is again cut into 4-5 sectors to initiate the sector culture.

Leaf Culture:-

Leaf culture is the culture of excised young leaf primordial or immature young leaf of the shoot apex in a chemically defined medium where they grow and follow the developmental sequences under controlled conditions.

Principle:-Leaf  primordial or very young leaves are excised, surface sterilized and inoculated on an agar solidified medium. In culture leaf remains in healthy condition for a long period. Leaves can be taken from aseptically grown plants for culture.Since leaves have a limited growth potential, so in culture the amount of leaf growth depends upon the stage of maturity at the time of excision.

Requirement:- Petri plates, Flasks, Scalpel, Young leaves, 5% Teepol, 70% ethanol, Sodium hypochlorite, 0.8% chlorine, D/W, MS media

Protocol:-

1)      Detach vegetative bud or very young leaf from shoot apex at the vegetative phase of the plant. Wash the explants thoroughly with running tap water.

2)     Immerse the leaf buds or young leaves in 5% Teepol for 10 min. Wash the explants to remove Teepol.

3)     Leaf  buds or young leaves are surface sterilization by immersion in 70% V/V ethanol for 30 sec. This treatment is followed by 10-15 min incubation in sodium hypochlorite solution with 0.8% available chlorine. Rinse the explants 3-4 times in sterile distilled water.

4)     Excise the leaf primordial from the leaf bud with help of surgical scalpel.

5)     Inoculate the leaf primordial or young leaf on to 20ml of solidified medium in a culture tube.

Anther Culture

Anther- A part of stamen containing pollen.

Culturing of anther obtained from unopened flower bud in the nutrient medium under aseptic condition.Callus tissue or embryoids from ather, that give rise to haploid plants though organogenesis or embryogenesis.

Principle:- The production of haploid plants is to exploit the totipotency of microspore.In this process the normal development and function of the pollen cell to become a male gamete is stopped and is diverted forcely to a new metabolic pathway for vegetative cell division.

Requiremnts:-Bud, MS medium,D/W,70% ethanol, Beakers, Flasks,Petridish

Protocol:-

1)Collection of unopened flower buds.

2)Surface sterilized with 70% ethanol.

3)Anthers excised from flower buds and kept separately.

4)Anthers in first meiotic division is selected by acetocarmine test.

5)Inoculated in the medium containing glutamine, L-serine and inositol.

6). Incubated the culture at 25C for 15 days. Here, anthers grow in to embryoids.

7)Embroyoids transfer to rooting medium under 300 lux illumination after 4-5 weeks the embryoids became plantlets.

8)For acclimatization, transfer to green house.