organ culture.pptx

Organ culture
Biotechnology
Horticulture

1. Organ culture Reema Naik M.Sc Horticulture (Fruit science)

2. Organ culture Organ culture can be defined as the organs or plant parts culturing in an artificial media or a culture from isolated medium. Any part of plant can serve as explants in organ culture-like shoot (shoot tip culture), root (for root tip culture), leaf (for leaf culture), and flower (for anther, ovary, ovule cultures). Most important types of organ culture for in vitro plant propogation are meristematic culture, shoot tip, nodal culture of separate lateral bud , isolated root, and embryo culture. The plant species can be rapidly propagated from small cuttings of the bulb by the technique of organ cultured bulb can be harvested after some period of culture in MS medium where growth rate, alkaloid, and microelements were reported to be many times (30-50) more in cultured bulbs.

3. Importance of organ culture i ) Organ culture provides an excellent exper­imental system to define the nutrients and growth factors normally received by the or­gan from other parts of the plant body and from its external environment. (ii) Organ culture is particularly valuable in studies of the interdependence of organs for growth hormones and other growth factors. (iii) Cultured organs may be ideally suited for studying specific problems in morphogen­esis.

4. Leaf culture Leaf culture is the culture of excised young leaf primordia 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 primordia 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. Leaf primordia or very young leaf have more growth potential than nearly mature leaves.

5. 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 minutes. Wash the explants to remove Teepol . 3) Leaf buds or young leaves are surface ster­ilized by immersion in 70% v/v Ethanol for 30 seconds. This treatment is followed by 10-15 minutes incubation in sodium hypo­chlorite solution with 0.8% available chlo­rine. Rinse the explants 3-4 times in sterile distilled water. (4) Excise the leaf primordia from the leaf bud with the help of surgical scalpel. (5) Inoculate the leaf primordia or young leaf onto 20 ml of solidified medium in a culture tube

6. 6) Incubate the culture at 25° C under 16 hrs. light.

7. Shoot tip culture/ meristem culture What is Shoot Tip Culture? Shoot tip culture may be described as the culture of terminal (0.1-1.0 mm) portion of a shoot comprising the meristem (0.05-0.1 mm) together with primordial and developing leaves and adjacent stem tissue. What is Meristem Culture? Meristem culture is the in vitro culture of a generally shiny special dome-like structure mea­suring less than 0.1 mm in length and only one or two pairs of the youngest leaf primordia, most often excised from the shoot apex.

8. Protocol Remove the young twigs from a healthy plant. Cut the tip (1 cm) portion of the twig. Surface sterilize the shoot apices by incuba­tion in a sodium hypochlorite solution (1% available chlorine) for 10 minutes. The ex- plants are thoroughly rinsed 4 times in ster­ile distilled water. Transfer each explants to a sterilized petri dish. Remove the outer leaves from each shoot apices with a pair of jeweler’s forceps. This lessens the possibility of cutting into the softer underlying tissues. After the removal of all outer leaves, the apex is exposed. Cut off the ultimate apex with the help of scalpel and transfer only those less than 1 mm in length to the sur­face of the agar medium or to the surface of filter-paper Bridge. Flame the neck of the culture tube before and after the transfer of the excised tips. Binocular dissecting mi­croscope can be used for cutting the true meristem or shoot tip perfectly.

9. Incubate the culture under 16hrs light at 25°C. As soon as the growing single leafy shoot or multiple shoots obtained from single shoot tip or meristem, develop root, transfer them to hormone free medium. The plantlets formed by this way are later transferred to pots containing compost and kept under greenhouse conditions 

10. Root culture Root culture can be defined as the culture of excised radical tips of aseptically germinated seeds in a liquid medium where they are induced to grow independently under controlled condi­tions.

11. 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  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.

12. Flower culture Flower culture can be defined as the aseptic culture of excised floral bud on a chemically de­fined nutrient medium where they continue their development to produce a full bloom in a culture vessel.

13. Protocol: (1) Flower buds or mature flowers are collected from the healthy plants. (2) Wash them thoroughly and dip them in 5% Teepol solution for 10 minutes and wash. (3) Transfer them to laminar air-flow cabinet. Surface sterilizes them by immersing in 5% Sodium hypochlorite, wash with autoclaved distilled water. (4) Using flamed forceps, transfer the flower bud or mature flower to culture tubes con­taining 20 ml solid medium. (5) Incubate the culture in 16 hrs. light at 25° C.

14. Ovule culture Ovule culture is an elegant experimental system by which ovules are aseptically isolated from the ovary and are grown aseptically on che­mically defined nutrient medium under controll­ed conditions. Importance of Ovule Culture: Ovule culture is a boon for the plant breeders in obtaining seedlings from crosses which are normally unsuccessful because of abortive embryos.

15. Embryo culture Embryo culture is a technique for cultivating an embryo under  aseptic condition on a nutrient medium. The method can be divided into two applications. One is performed with mature embryos and helps mainly in shortening the period of germination by overcoming seed dormanc y . The other is performed with immature embryos and is called early embryo rescue.