In this article we will discuss about the Embryo Culture:- 1. Meaning of Embryo Culture 2. Methods of Embryo Culture 3. Nutrient Medium 4. Factors 5. Significance.
Meaning of Embryo Culture:
The method of culturing embryo excised from the seed on sterilized culture medium is called embryo culture. Haning in 1904 working with Raphanus and Cochlearia (Brassicaceae) first successfully cultured embryos ‘in vitro’. Later various workers cultured embryos of different plants.
Dietrich (’25) used semi-solid medium containing some minerals, 2.5—5% sucrose, 1.5% agar for embryo culture. In such a medium mature embryos grew well, but immature embryos did not follow normal developmental stages. Such embryos skipped certain developmental stages and grew directly into seedlings.
Laibach (’25, ’29) successfully applied embryo culture technique for raising plants from very light, greatly shrunken hybrid seeds, which normally fail to germinate. He worked with interspecific crosses of Linum ‘(Linum austriacum X L. pererme). He cultured the excised embryos from hybrid seeds on moist blotting paper or cotton wadding containing 10—15% sugar or glucose and obtained fertile hybrid plants.
This technique has been widely used to raise hybrid plants from abortive embryos of different interspecific crosses of various plants (such as, Hordeum, Linum, Prunus etc.) Jorgensen (’28) used this method to raise hybrids of Solanum nigrum xS. luteum. Beasley (’40) used it for raising hybrids of Gossypium hirsutumxG. herbaceum. Smith (’44) used this technique in crosses between Lycopersicon esculentum x L. peruvianum.
In interspecific crosses of Prunus, Lilium and Datura this method was used. In interspecific crosses of Prunus (between species, such as, Prunus avium, P. domestica, P. persica etc.) if early varieties are used as female parents then the seeds abort. Takey (’44) cultured excised embryos of such Seeds and obtained healthy, fertile plants.
Sapre (’63) cultured embryos of interspecific hybrids of Oiyza on Nitsch’s medium and obtained transplantable plants. Iyer and Govila (’64) cultured embryos of various interspecific hybrids of Oryza on semi-solid medium supplemented with 10% coconut milk, 0.1% malt extract and obtained seedlings which showed almost 100% survival.
Sometimes embryos of interspecific crosses may die at later stages of seed development due to failure of endosperm development or due to incompatibility between embryo and endosperm. Cooper (’78) obtained hybrid plants between barley and rye by embryo culture.
Sometimes due to chromosome elimination haploid embryo is formed and endosperm degenerates. Such embryos may be excised and cultured successfully in vitro and from this haploid plantlets are obtained.
Methods of Embryo Culture:
For culturing the embryos under aseptic conditions several measures are adopted. Working area is sterilised. Scalpels, needles, forceps etc. are dipped in 70% alcohol and then flamed. The selected fruit is surface sterilised for 15 minutes with alcohol or calcium hypochlorite or sodium hypochlorite solution and then washed thoroughly in double distilled water.
The fruit is cut and ovule or seed is taken out with a sterile forceps and placed on a sterile petridish. The seed or ovule is cut open and the embryo is extracted. The excised embryo is immediately placed on the nutrient medium of a culture vial under aseptic conditions.
Planted embryos on tubes, flasks, bottles or other containers are kept at room temperature in diffused light. Seedlings are produced and these are transferred to a sterile growth stimulating medium before transfer to the field conditions.
Nutrient Medium of Embryo Culture:
The nutritional requirement of a growing embryo grad- dually decreases and its biosynthetic capacity gradually increases. So the composition of the culture medium will depend upon the stage of the embryo to be cultured.
For culturing young embryos (heteromorphic phase) inorganic medium only is not sufficient. Such a medium should be supplemented with organic substances, such as, yeast extract, malt extract, sugar etc. La Rue (’36) used IAA instead of yeast extract.
In 1942 Van Over-beck used coconut milk in culture medium. In 1944 Blakeslee and Satina used malt extract. Sander (’48, ’50) said that various plants have different nutritional requirements.
In jute many attempts to raise interspecific hybrids of Corchorus olitorius X C. capsularis failed due to post fertilization barriers. Islam (’64) used a medium supplemented with 0.1% yeast extract, 0.05 ppm kinetin, 0.05 ppm IIA for culturing the hybrid embryos of the cross between C. capsularis (♀) x C. olitorius (♂). In this medium the hybrid embryo grew into transplantable seedling.
It is easier to culture differentiated embryos. Such embryos can grow on culture medium containing some mineral salts and sucrose only, as they can synthesise most of the substances required for their growth. This stage is called autotrophic phase.
Factors Influencing Embryo Culture:
(1) Stage of the embryo:
Globular or post-globular embryos are cultured on medium supplemented with coconut milk, yeast extract or casein hydrolysate or growth hormones (such as auxin, cytokinin, gibberellin etc.).
Preglobular embryos usually fail to grow in culture or form unorganised callus tissue. This suggests that the mother tissue adjacent to the embryo not only supplies nourishment to the young embryo, but also helps the young embryos to pass through a predetermined course of embryogenesis.
Young embryo of Cuscuta reflexa and globular embryo of Dendropthoe fulcata on culture produce callus tissue which later form many embryoids (Fig. 13). In barley Norstog (’65) observed that pro-embryos measuring 0.1 mm or less fail to grow normally. They produce callus tissue from which embryoids are formed. But he noted that larger embryos (0.15 mm) of barley can grow in normal culture medium.
(2) Pre-treatment:
In certain cases if embryos are chilled before culture then better response of embryos on culture has been noticed. In Prunus, if the embryos are stored at 5°G for one month or at 1°G for 4 months then they grow better.
(3) Endosperm:
Influence of endosperm in embryo development is significant. Krause (’74) observed that hybrid embryos from a cross between Hordeum and Secale survive on culture only in 1 % cases.
But such an embryo on culture when placed on an endosperm, then the viability increases to 30—40%. This indicates that the young embryos in nature get their required growth substances from the endosperm and adjacent maternal tissue.
De Latour (’78), Williams (’78, ’80) and Williams and De Latour (’80) developed methods of culturing young hybrid embryos in excised endosperm from normal ovules of either parent or of other species.
Using this method they were able to raise several interspecific hybrids of Trifolium. In Colocasia esculenta embryos fail to develop due to inadequate food supply from the endosperm. In 1960 Abraham and Ramachandaran excised the embryos and grew them on culture and obtained seedlings.
Regeneration potentiality of different parts of an embryo:
Regeneration capacity of different parts of an embryo varies. Lee (’55) working with lupin noted that those segments which included the plumule can form seedlings. Bajaj (’66) observed similar behaviour of the embryo in Dendropthoe fulcata. Cotyledons play an important role in the development of the seedling.
It has been observed in Avena, Hordeum, Pennisetum etc. that if part of the cotyledon is removed before culture then the development of the embryo is suppressed according to the size of the removed cotyledon. In Cassythea radicular halves of the cotyledon control shoot development.
If the plumular halves of both cotyledons are removed then also healthy shoots can develop. (Fig. 14). In 1981 Tilton observed in Zea mays that presence of scutellum is essential for growth and development of roots.
Culture of embryos of stem and root parasites in absence of hosts:
In 1963 Rangaswamy and Rangan successfully cultured the embryos of Cassytha filiformis, a stem parasite in absence of host or its stimulus.
In Orobanche egyptica, a root parasite culture of seeds with unorganised embryos produces callus tissue which later differentiates into shoot tip’s. Similar observations were made by Rangan in 1965 working with Cistanche tubulosa.
Significance of Embryo Culture:
(1) We can study the nutritional requirements of the embryos at various developmental stages by embryo culture.
Differentiated embryos can grow in a simple medium containing sucrose and few mineral salts (autotrophic phase), whereas young embryos cannot grow on such a simple medium (heterotrophic phase).
(2) By embryo culture it is possible to know the factors controlling differentiation of embryos.
(3) Many interspecific hybrid plants can be produced by embryo culture. Utilizing this technique wider range of hybridization can be done with success.
Many desirable crosses are not possible due to some pre-fertilization barriers or poor and abnormal endosperm development or endosperm-embryo incompatibility. In cross between Hordeum vulgare X H. bulbosum no endosperm is formed and the embryo aborts.
Davis (’60) successfully cultured the embryos excised 14—28 days after pollination and raised the seedlings. In cross involving Lirtum auratum and L. speciosum-album due to embryo-endosperm incompatibility seedlings are not formed. By culturing young embryos Bhatnagar and Bhojwani produced hybrid plants.
(4) Shortening of dormancy period:
By embryo culture the dormancy period of seeds can be shortened. In Iris seeds remain dormant for 2-3 years, but by embryo culture such seeds can germinate in the same year.
Nickel (’51) observed that Malus seeds take about 9 months to germinate. But the excised embryos on culture started germination within 48 hours and within 4 weeks produced transplantable seedlings which grew to a height of one meter approximately within five months.
(5) Propagation of rare plants:
Some abnormal coconuts instead of producing endosperm form solid soft tissue, such nuts are rare and costly and are called ‘makapuno’. The seeds of this variety failed to germinate normally. In 1969 de Guzman was successful in producing makapuno seedlings by using embryo culture technique.
(6) Seed viability test:
According to Barton (’61) by embryo culture we can know about seed viability.
(7) Production of haploids:
From haploid plants arising from crosses between distantly related plants viable plants can be produced by embryo culture.
(8) Introduction of disease resistance character:
For introducing disease resistance character from wild tomato Lycopersicon peruviartum to cultivated tomato L. esculentum crosses were made but no seedling is obtained. Bhojowani and Bhatnagar cultured hybrid embryos excised thirty-five days after pollination and obtained callus followed by plantlet formation.