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Embryo culture

Embryo culture involves the aseptic isolation and in vitro culture of embryos from seeds or ovules under controlled conditions to promote plantlet development. Various categories of embryo culture include mature, surgically dissected, immature embryos, and cultures from polyembryonic seeds or inviable embryos. The method plays a significant role in studying embryonic growth, overcoming seed dormancy, and understanding plant developmental processes.

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EMBRYO CULTURE EMBRYO CULTURE :- "The embryo of different developmental stages, formed within the female gametophyte through sexual process, can be isolated aseptically from the bulk of maternal tissues of ovule, seed or capsule and cultured in vitro under aseptic and controlled physical conditions in glass vials containing nutrient solid or liquid medium to grow directly into plantlet." DIFFERENT CATEGORIES OF EMBRYO CULTURE:- 1.Culture of mature and intact seed embryo :- The aim of this embryo culture is to analyse the var- ious parameters of embryonic growth and the metabolic and biochemical aspects of dormancy and germination.
2. Culture of surgically dissected embryos:- The mature seed embryo can be dissected surgically into a number of segments. Such embryo segments are cultured to analyse the rela- - tionship of different parts of the embryo to its final form in culture. 3.Culture of immature embryos or proembryos:- Globular and heart-shaped stages of embryo are appropriately called as pro- embryos. The objective of such culture is to understand the control of differentiation and the nutritional requirements of such progressively developing embryos. 4. Culture of intact seed containing undifferentiated embryo:- Each fruit of an orchid plant develops sev- eral thousand tiny seeds which contain morpho- logically undifferentiated embryos. These em- bryos are the spherical mass of tissue lacking both radicle and plumule. There is even no stor- age tissue in the seeds and the seed coat is re- duced to a membranous structure. For this reason, the entire seed of orchid containing undifferentiated embryo is cultured and treated as embryo culture.
5. Culture of adventive embryo from polyemryonic seed:- Besides the zygotic embryo produced from egg cell, some additional embryos are produced from nucellar tissue in polyembryonic seed like lemons and oranges. Such additional abortive embryos can be exploited in culture for clonal propagation. 6. Culture of inviable or abortive embryo:- In many inter-specific or intergeneric breed- ing experiments, sometimes inviable or abortive embryos may develop due to unsuccessful cross- es. As a result, the non-viable seeds do not ger- minate normally. But it is now possible to raise a hybrid plant by culturing the inviable embryos in vitro. PRINCIPLE OF EMBRYO CULTURE:- The underlying principle of the method is the aseptic excision of the embryo and its transfer to a suitable nutrient medium for development under optimum culture conditions. PROTOCOL:-
The following protocol for embryo culture is based on the method used for Capsella bursa-pastoris. With modification, this basic protocol should be applicable to embryo culture in general. STEPS :- 1. Capsules (dry fruit or a capsule is a structure composed of two or more carpels.) in the desired stages of development are surface sterilized for 5-10 minutes in 0.1% HgCl2, either in a closed small room previously illuminated by UV lamps or in a Laminar air flow. 2 . Further operations are carried out under a specially designed dissecting microscope at a magnification of about 90X. The capsules are kept in a depression slide containing few drops of liquid medium. 4. The outer wall of capsule is removed by a cut in the region of the placenta; the halves are pushed apart with forceps to expose the ovules. 5. A small incision ( micropyle) in the ovule followed by slight pressure with a blunt needle to free the embryos. 6. The excised embryos are transferred by mi- cropipettes or small spoon headed spatula to standard
10 cm petridishes containing 25 ml of solidified standard medium. Usually 6-8 embryos are cultured in a petridish. 7. The pertridishes are sealed with cellotape to prevent desication of the culture. 8. The cultures are kept in a culture room at 25± 1°C and given 16 hrs. illumination by cool white fluorescent tube. 9. Subcultures into fresh medium are made at approximately four weeks intervCULTURE ( note :- for fresh seeds or dry and imbibed seeds the schedule is slightly changed. 1. Seeds are cleaned by 5% Teepol (a liquid detergent) for 10 minutes 2. dipped in 70% ethyl alcohol for 60 seconds. 3. Surface sterilization in 0.1% HgCl, is followed by washing in sterile water. 4. Then the seeds are decotylated using a sharp scalpel and embryos are transferred to solid nutrient medium. ROLE OF SUSPENSOR IN EMBRYO CULTURE:- )
The suspensor is traditionally believed to be a supporting structure during plant embryo development that pushes the embryo proper into the endosperm cavity and connects it to the surrounding maternal and endosperm tissues to facilitate the transfer of nutrients and plant hormones. APPLICATION OF EMBRYO CULTURE IN PLANT SCIENCE:- Importance of embryo culture in relation to biological knowledge:- • It helps determining the factors that regulate the growth of the primodial organs of the seedling plant. • It helps to study the metabolic and biochemical aspects of dormancy and germi- nation. • It helps in analysis of various parameters of embryonic growth. • The culture of proembryos helps to understand the control of differentiation and the nutritional requirements of progressively smaller embryos. • The culture of surgically dissected embryos segments has facilitated understanding the
relationship of the different parts of the em- bryo to its final form in culture. Applied aspect of embryo culture:- ❖ Culture of non- viable or abortive embryo ❖ To overcome seed dormancy ❖ To shortening the breeding cycle ❖ To overcome self sterility of seeds ❖ Seed testing. Other applications:- ❖ To study the evolutionary relationship. ❖ To study the host parasitic interaction. ❖ To study the mutagenic effect.