• TOPIC: • TRIPLOID PRODUCTION (ENDOSPERM

CULTURE)- DIFFERENTIATION AND FACTORS AFFECTING THEM- APPLICATIONS

• SOMATIC EMBRYOGENESIS- FACTORS AFFECTING, STAGES OF SOMATIC EMBRYOGENESIS, LARGE SCALE PRODUCTION, SYNTHETIC SEEDS CONCEPT AND ITS PRO’S AND CON’S

PRESENTED BY ZUBY GOHAR ANSARI TAM/14-26

ENDOSPERM CULTURE (HISTORY AND DEF.) • Double fertilization occurs in majority of

angiosperms which results in two fusion products i.e. Zygote (fusion product of egg and one of the male gamete) and triploid endosperm (fusion product of polar nuclei and second male gamete).

• The endosperm lack many organogenic potential and vascular differentiation.

• Endosperm is unique tissue, firstly in its function of supplying nutrition to developing embryo and secondly in being triploid.

• Attempts to grow endosperm tissue in cultures began in 1930’s and now mature and immature endosperm of various taxa of angiosperm have been grown.

• Lampe and Mills (1933) : They were first to report the proliferation of immature endosperm tissue of maize grown on medium containing extract of potato.

• Nakano et al. (1975): Successfully cultured immature endosperm of rice and achieved organogenesis.

• Rangaswami and Rao (1963): Successfully cultured mature endosperm of santalum album (chandan) up to callus.

• Endosperm culture: It is the in vitro development of isolated mature or immature endosperm from seed at proper stage on a suitable culture medium to obtain triploid plantlet.

TYPES OF ENDOSPERM CULTURE• 1. Mature endosperm culture: It is the in vitro

development of isolated mature endosperm from ripen endospermic seed on suitable culture medium to obtain triploid plantlet is called mature endosperm culture.

• 2. Immature endosperm culture: It is the in vitro development of isolated immature endosperm isolated at precise stage from immature seed, cultured on suitable culture medium to obtain triploid plantlet is called immature endosperm culture.

STEPS IN ENDOSPERM CULTURE• It consist of mainly 3 steps:• 1. The immature or mature seeds are dissected

under aseptic conditions and endosperms along with embryos are excised.

• 2. Excised endosperms are cultured on a suitable medium and embryos are removed after initial stage.

• 3. Callus followed by embryogenesis or shoot bud differentiation.

• 4. Complete plant formation.

PROCEDURE OF ENDOSPERM CULTURE• 1. Explant source: In most of the cereals, mature

endosperm does not respond to cultural conditions. It is therefore, excised at proper period of development. Normally the endosperm of cereals undergo certain changes 12 days after pollination making this able to respond. Proper stage for maize is 8-11 days after pollination, 4-7 days after pollination for rice, and 8 days after pollination for wheat.

• In some families association of embryo tissue in initial stages seems essential for proliferation of mature endosperm. In such cases, entire seeds are used as explant. Immature seeds provide explant for non-endospermic seeds.

• 2. Inoculation of explant: For in vitro culture of mature endosperm, seeds having massive endosperm are decoated, surface sterilized with suitable disinfectant and after 2-3 washings with sterile distilled water, planted on the nutrient medium. For in vitro culture of an immature endosperm, the entire seed or kernel is surface sterilized and the endosperm tissue is excised under aseptic conditions. In cereals, top of kernel or immature ovaries (micropyle end) are cut off with a sterile knife and exposed endosperm squeezed out and placed on the callus induction nutrient medium.

• The basal white and MS medium is generally used to induce callus from an immature endosperm.

• The basal medium is supplemented with tomato juice, yeast extract, grape juice. Sucrose (2-4%) is used as a source of carbohydrates. In some cases, addition of 2,4-D or IAA, cytokinin necessary.

• 3. Incubation/ maintenance of culture: To induce callus, the endosperm cultures are maintained darkness or diffused light. Differentiation take place when calli are transferred to bright light (2000 lux – 4000 lux) and 25⁰C temperature

• 4. Shoot bud differentiation or embryogenesis: Plantlet formation from endosperm callus maturation follow organogenic or embryogenic mode of development. Embryo differentiation occurs when the proliferated tissue is transferred from callusing to basal medium with or without gibberellins. So culturing carried out up to complete development of plantlet.

• 5. plantlet developed are hardened in green house by transferring in vermiculture media and maintaining proper humidity.

FACTORS AFFECTING ENDOSPERM CULTURE • 1. Explant stage: proper stage may vary from

cellular (immature) to mature endosperm depending upon species. (4-7 DAP in rice, 8 DAP in wheat and 12 DAP in maize and fully matured in santalum album).

• 2. Nutrient medium: Low amount of reduced nitrogen is required for proliferation. Undefined source like tomato juice, yeast extract, etc.

• 3. Physical factors: The pH 7.0 seems to be effective for fresh weight increase. Maximum growth of endosperm occurs between 24-27⁰C temperature and 12-16 hours photoperiod with diffuse day light supported callusing as well as regeneration.

• 4. Embryo factors: association of embryo tissue in the initial stages seems essential for inducing proliferation of mature endosperm tissue in some families. Immature seeds of non endospermic seeds exhibit no dependence on embryo factor.

APPLICATIONS OF ENDOSPERM CULTURE • Techniques of endosperm culture has enabled

the production of triploid plants. Triploid plants are self sterile and usually seedless. The trait increases edibility of fruits and is desirable in plants such as apple, banana, grape, watermelon and mango which are commercially important.

• In timber and fuel yielding plants, triploids show better performance over their relative diploids or tetraploids. Also there is no problem of seed sterility as they can be multiplied by vegetative means.

SOMATIC EMBRYOGENESIS• Regeneration of embryos from somatic cells, tissues

or organs either denovo or directly in vitro conditions is known as somatic embryogenesis.

• It is also known as non- zygotic/ non-sexual embryogenesis.

• Various terms for non-zygotic embryos have been reported such as,

• A. Adventive embryos: Somatic embryos arising directly from other organs or embryos.

• B. Parthenogenic embryos: Formed by unfertilized eggs.

• C. Androgenetic embryos: Formed by male gametophyte.

• Occurence of asexual embryogenesis is generally restricted to intraocular tissues.

• Differences between somatic and sexual embryos

SOMATIC EMBRYOS SEXUAL EMBRYOS

Embryos arises from single cell. Arises from multi cell.

Embryos have bipolar structure. It is monopolar structure.

Embryos has no vascular connections with cultured explant.

Embryos has vascular connections with cultured explant.

Induction of somatic embryogenesis requires single hormonal signal.

Requires two hormonal signals.

• The initiation and development of embryos from somatic tissues in plant culture was first recognized by steward et al (1958) and Reinert (1958- 59) in cultures of Daucus carota (carrot).

• In addition to the development of somatic embryos from sporophytic cells, embryos have been obtained from generative cells, such as the classic works done by Guha and Maheswari (1964), with Datura innoxia microspores.

TWO ROUTES TO SOMATIC EMBRYOGENESIS

• Sharp et al (1980) divided 2 routes to somatic embryogenesis.

• 1. Direct embryogenesis: The embryo initiates directly from the explant tissue in absence of callus proliferation.

• This occurs through pre-embryonic determined cells (PEDC). Such cells are found in embryonic tissues.

• Eg: scutellum of cereals

• 2.Indirect embryogenesis: The embryos initiate from the explant through callus proliferation.

• This occurs through Induced embryogenic determined cells (IEDC).

• Eg: Secondary phloem of carrot, leaf tissue of coffee, petunia, asparagus etc.

• For some species, any part of plant body serves as an explant, for embryogenesis. But in some species only certain regions of explant may respond in culture. Eg: cereals

• Floral or reproductive tissue in general has proven to be an excellent source of embryogenic material.

• The physiological states of the plant from which explant is taken is also extremely important.

STAGES OF SOMATIC EMBRYOGENESIS

• Somatic embryogenesis encompasses various stages such as

• 1. Callus initiation

• 2. Embryo development and maturation

• 3. Plantlet formation

FACTORS AFFECTING SOMATIC EMBRYOGENESIS

• 1. Genotype of explants: Explant genotype has a marked influence on somatic embryo regeneration and in many cases, it may determine whether or not somatic embryo regeneration will occur.

• Strong genotypic effect have been shown in many species.

• Eg: Alfa-alfa , wheat, maize, rice and chick pea etc.

• 2. Growth regulators: • Auxins: Auxin alone or in combination with cytokinin

appear essential for the onset of growth and the induction of embryogenesis of all the auxins, 2,4-D followed by NAA have proven to be extremely useful.

• Effective concentration ranges are 0.5 – 27.6 μ M for 2,4-D and 0.5 – 10.7 μ M for NAA.

• Cytokinins: CKs have been used in the primary medium invariably during embryogenesis of crop plants.

• Effective concentrations for kinetin0.5 – 50.0μ M. CKs are important in fastening somatic embryo maturation and especially cotyledon development.

• ABA: It is added at inhibitory levels @0.1- 1 μM promotes somatic embryo development and maturation and at same time inhibits abnormal proliferation and initiation of accessory embryos.

• 3. Nitrogen source: Form of nitrogen has marked influenced on somatic embryogenesis. In carrot NH₄⁺ form has a promotive effect.

• Somatic embryos development occurs on a medium containing NO₃⁻ as the sole nitrogen source.

• 4. Other factors: High K⁺ levels and low dissolved O₂ levels prevents somatic embryo regeneration.

• In citrus medica, volatile compounds like ethanol, inhibit somatic embryo regeneration.

• In soybean low sucrose conc. @ 5 to 10 g/l promotes somatic embryogenesis than high concentration.

• In Alfa-alfa, use of maltose as a carbon source improved both somatic embryogenesis induction and maturation as compared to sucrose.

SYNTHETIC SEED CONCEPT• Synthetic seeds are also known as artificial seeds

and also encapsulated embryos.• The aim of somatic embryo encapsulation is to

produce an analog to true seeds, which is based on the similarity of somatic embryo with zygotic embryo with respect to their morphology, physiology and biochemistry.

• Two type of synthetic seeds have been developed namely,

• 1. Hydrated S.S.• 2. Dessicated S.S.

• 1. Hydrated synthetic seeds: Renderbergh et al (1986) developed hydrated artificial seeds by mixing somatic embryos with an encapsulation matrix so that embryos are well protected in that matrix and it is rigid enough to allow for rough handling.

• Encapsulation matrix consists of hydrogels such as agar, carrageenan, alginate or plant exudates of arabica, karaya or seed gums of guar, tamarind or microbial products like dextran, xantham gum and divalent salts.

• Various divalent salts which can be used are CaCl₂, Ca(OH)₂, ferrous chloride, cobaltous chloride etc.

• Sodium alginate + CaCl₂ = encapsulation of somatic embryo.

• Procedure: Mixing the somatic embryos with sodium alginate, followed by dropping into a solution of CaCl₂, CaNO₃ to form calcium alginate beads.

• Calcium alginate capsules tend to stick together and are difficult to handle because they lose water rapidly and dry down to a hard pellet within a few hours of exposure to the atmosphere.

• These problems can be offset by coating capsules with Elvax 4260 (Ethylene vinyl acetate acrylic acid tetrapolymer)

• Sodium alginate + CaCl₂= Calcium alginate

• 2. Dessicated synthetic seeds: Kim and Janic (1989)applied synthetic seed coats to clumps of carrot somatic embryo to develop dessicated artificial seeds.

• Mixing of equal volume of embryo suspension and 5% of polyethylene oxide (a water soluble resin), which subsequently dried to form polyembryonic dessicated seeds.

• The survival of encapsulated embryos was further achieved by embryo hardening treatments with 12% sucrose or 10⁻⁶ M ABA, followed by chilling at high inoculum density.

• Potential uses of synthetic seeds: • Produce large number of identical embryos .• Determines the roles of endosperm in embryo

development and germination.• Propagation with low cost, high volume

capabilities of seed propagation.• Produced within short time and in any season,

at any time.• Dormancy period can be reduced there by

shortening the life cycle of a plant.• Useful in preserving germplasm.

• Provides knowledge to understand the developmental, anatomical characteristics of endosperm and seed coat formation.

• Synthetic seeds production has been successfully obtained in Zea mays, Opium, Daucus carota, Lactuca sativa, Madicago, Brassica species, Gossypium, Santalum species etc.

• Disadvantages with artificial seeds: • 1. Facilities required are costly.• 2. Special skills are required.• 3. Errors in maintenance.• 4. Specific kinds of genetic and epigenetic

modifications can develop.

• Problems in production of synthetic seeds:• 1. Artificial seeds that are stable for several months

requires the procedures for making embryos quiscent.

• 2. Synthetic seeds need to be protected against dessication.

• 3. Recovery of plants is often very low due to incomplete embryo formation or difficulties in creating artificial endosperm.

• 4. Embryo must be protected against microbes.