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Zygotic Embryo Culture

IntroductionGroup Members:

Maisa Faria 1236Mohoshina Hossain 1234Abida Sultana 1245MD. Shariful Islam 1276Najia Sultana 2077Shahariar Hossain 2228MD. Iftekhar Hossain Dipto 2319MD. Ariful Islam Sagar 2340Arman-UI Hoque 2344Hasan Ali 2554

Zygotic Embryo Culture

Introduction

Zygotic embryo is formed following double fertilization of the ovule, forming the plant and the endosperm to gather go into the seed.

Zygotic embryo culture is the aseptic isolation and growth of sexually produce embryo in vitro with the objectives of obtaining viable plant.

History

• Embryo culture in the field was provided by Laibachi.

• In 1904,Hanning published a paper describing the first systematic attempt to culture isolated mature embryos of angiosperm.

• Laibachi(1925,1929) cultured excised embryo from seeds of an interspecific cross Linum perenne cross L.austrainum and succeded in raising hybrid plant.

Types of embryo culture

According to Pierik (1989) there are in principle 2 types of embryo culture-• Culture of immature embryo:

This type of embryo culture in mainly used to grow immature embryos originating from unripe or hybrids seeds which tail to germinate.

The chance of success in this type of culture depend largely on the development stage of the excised embryo.• Culture of mature embryo:

Mature embryo are excised from ripe seeds and cultured mainly to avoid inhibition in the seed for germination.

Fig: Types of embryo

Important aspect for technique:

Two most important aspect of zygotic embryo is

1. Composition of the culture medium2. Excision of the embryo

Technique: The choice of plant material may become important when the objective is to introduce the technique to beginner.

Plant Material: • Embryo which can be easily dissected out should

select.• Mature embryo of seed legumes and crucifers

possessing a large seeds are good starting material.• At the same stage of development large number of

genetically uniform embryos can be obtained.

Surface Disinfection: Disinfection of embryo surface is unnecessary unless a systematic infection is present.Instead mature seeds entire ovules or fruits are surface sterilized and embryo removed aseptically from the surrounding tissue.

Sterilization: • Entire capsules are surface sterilized and seeds removal

under aseptic conditions.

• There are then spread in a single layer on the surface of an agar medium using a sterile needle.

• Sterilization is carried out by immersing the material in hypo chloride.

• Containing commercial bleach (5-10% clorox,0.45% Caocl2 or NaOCl) for 10-5 minute or ethanol (70-75) for 5 minute.

• A small amount(0.01-0.1)% of a surfactant (Tween 20, Tween 80, Teepol or Mannoxol) added to the disinfection solution to increase the tissue wettability.

Excision of embryo: For the in vitro culture of embryos generally it is necessary to free them from their surrounding tissues.• In case of dicot, the procedure of excision embryo is given

below-• Keep disinfected capsules in a few drops of the sterile

culture medium.• Remove outer walls by an incision in the region of the

placenta and pull two halves apart with forceps to expose ovules.

• Detach a single ovule from the placenta and place in the depression (cavity) of a new sterilized slide containing a drop of medium.

• Split the ovule longitudinally using a shape mounted blade.

• Carefully tease apart the ovule tissue in order to free the entire embryo along with the attached suspensor.

• To excise older embryo make side lacking the embryo apply pressure with a blust needle to set the embryo free.

Fig: Isolation of embryo (monocote)

In case of monocot the procedure of isolation embryo is given below-• Rinse the disinfected caryopsis with sterile water.

• Place it in a sterile Petridis with the rachilla underneath.

• Remove fruit wall and seed coat.

• Carefully isolate the entire embryo with plumule, scutellum and radical.

Embryo-nurse endosperm transplant: The endosperm transplant technique for culturing young (immature) embryos is explained in figure. Williams and De Lautour inserted an excised hybrid embryo into a normally developing ovule of ones of the parents or a third species and cultured the nurse endosperm with the transplanted embryo on the nutrient medium.

Nutritional Requirement: The nutritional requirement of an embryo during its development in vivo constitute 2 phases-

• Heterotrophic phase- An early phase where in the embryo in dependent and draws upon the endosperm and material tissue.

• Autotrophic phase- A later phase in which the embryo in metabolically required for synthesizing substances required fairly independent for nutrition.

Media constituents for in vitro of young or immature embryos also differ from those od mature embryo.

Monnier (1976) developed a unique method which allow complete development of younger embryo (early globular stage) up to germination without moving them from their original position in culture.

The Composition Of Media:

Mineral Salts :

• Higher levels of Potassium (by adding 350 mgl-1)

• Ca ( Double concentration of Cacl2 )

• Reduce level of NH4NO3 & FeEDTA

• Double concentration of MS micro nutrient

Carbohydrate :

• Sucrose is most commonly used in embryo culture

• Glucose & Sucrose maintain osmolarity of the culture medium which is critical with respect to the age of embryo

• Mature embryo grow fairly well at 2% low concentration of sucrose but younger embryo at higher level of carbohydrate

• Various concentration of sucrose used in embryo culture depend on the species and a size / age of embryo

Nitrogen & Vitamin :

• NH4NO3 is significantly superior to KNO3

• NaNO3 & (NH4)2 HPO4

• The presence of NH4+ in the medium has been found essential for proper

growth & differentiation of embryo

• Aspargine & Glutamin is the superior source of Nitrogen

Natural Plant Extract :

• Coconut Milk • Water extract of dates• Water extract of bananas • Hydrolyzation of wheat gluten• Tomato juice• Alcohol diffuseness of young seeds of Irish, Lupinus, Datura, etc.

Growth Regulator :

• Auxin & Cytokine are not used in embryo culture because they induce callus formation

• GA3, ABA is generally used as growth regulator

PH : 5 - 7.5

Incubation Condition :

• Temperature : 1. Embryo culture shows a favorable response at elevated temperature (27-30 °c)2. Incubation temperature to culture embryos of species ( Brassica hybrids)

occurring in cold region or season range from 17-22 °c (see Hu & Wang 1986)

• Light : A type of recalcitrant secondary dormancy is induced when these embryos are excised to 4000 lx or more for a 4 hour period during the initial 4 days of incubation.

Suspensor in embryo culture

• The suspensor is ephemeral structure found at the radicular end of the pro-embryo and attained maximum development.

• Without it embryo culture is difficult

• Older embryo grow well with or without suspensor

• The presence of suspensor is critical particularly for the survival of young embryo

• It may be substituted by the addition GA or ABA to the culture medium

Precocious Germination

• According to Walbot (1978) embryo development is classified into 5 stages

• Excised immature plant embryo on a nutrient medium tend to bypass the stage of dormancy and cease to undergo linear embryogenic development. The embryo developed instead into weak seedlings. The phenomenon of seedling formation without completing normal embryogenic development is called precocious germination.

Morphogenesis in culture of seeds with partially differentiated embryo :

• An unorganized embryo where seedlings arise without under going further embryogenic differentiation

• The embryonal & Proximal to the micropyle is regarded as the radicular pole & distal to the micropyle as the plumular pole

• In monopolar pattern only one of the pole is involved in the development of seedlings

• Plumular pole enlarges to form a spherule like structure called “ Protocorm ” after turning green attain certain size differentiates roots & shoots

• Sometimes radicular pole of the given rise to radicular cylinder whose tip penetrate the root of host while the portion of the cylinder remain outside the proliferates into an irregular mass of tissue called tubercle from which shoot differentiate.

Morphogenesis in culture of seeds with partially differentiated embryo :

• Monopolar pattern of seedlings development can be modified by media composition in by polar pattern TB medium CM or yeast extract IAA (0.1 mgl-1) Kinetin ( 0.5-10 mgl-1) GA3 (0.5-30 mgl-1) Strigol (0.01μgl-1)

• In bipolar pattern plumule pole differentiates shoot bud & ridiculer pole roots• Glucose, mannose or raffinose favored bipolar germination

Organogenenic potential of embryo callus :

• An embryo callus is reported to possess a high regenerative capacity compared to those derived from mature organs such as - leaf, stem & roots

• No differentiation occurred if the callus originate from a mature embryo • Immature embryo are good explants for initiating callus capable of plant regeneration.

Example – Oats, Barley etc• To obtain Callus with morphogenic potential excised immature embryo are placed on

an agar medium with the scutellum facing up in the presence of 2,4-D alone or in combination with cytokinin

Factors Affection Zygotic Embryo Culture :• Nutritional requirement : It is vary at different stage of embryo

• Carbohydrate : Sucrose is most commonly used in embryo culture

• Natural Plant extract : Coconut milk is an important factor

• Nitrogen & Vitamin : CH is a very important factor which has it’s optimum level (50-500mgl)

• Growth regulator : 0.01mgl-1GA promotes embryogenesis of young barley embryo without inducing precautious germination

• Physical factor : • PH : 5-7.5• Light : 4000 lux for 4 hour period

Application of Zygotic Embryo Culture :• Obtaining rare hybrids : Crossing cultivated tomato

(Lycorpersicon esculentum) with wild tomato (L. peruvianum) has been considered describable from the point of vies of transferring pests & diseases resistance from the later to the former. The cross does not succeed dew to pre-fertilization barriers. In the reciprocal cross however fertilization occurs but the cross fails because of embryo absorption. But by the aid of embryo culture hybrid plants can be produced from these two species.

The embryo callus culture approach has also yielded hybrids from the crosses Lycopersicon esculentum × Solanum hycopersicoides in which embryos capable of direct plant formation do not developed.

Application of Zygotic Embryo Culture• Haploid Plant Production: In the cross of Hordeum vulgare ×

H. bulbosum fertilization proceeds readily but the chromosome of H. bulbosum are preferentially lost during the first few divisions of embryogenesis. As a result the haploid embryos show slow growth. This coupled with the disintegration of the endosperm 2-5 days. After fertilization necessitates the culture of the excised embryo to raise the haploid Hordeum vulgare plants.

• Overcoming dormancy & shortening the breeding cycle : Occasionally the breeding on horticultural plants such as – deciduous plants is delayed dew to long dormancy period of their seeds. By growing excised embryos in nutrient medium this period may be reduced. For example - using embryo culture (Randolph & Cox 1943) could shorten the lifecycle of Irish from 2/3 years less than 1 year.

Application of Zygotic Embryo Culture• Rapid seed viability test: The possibility of breaking seed

dormancy by embryo culture also allows the use of this technique for rapid testing of the viability of a particular batch of seeds. Germination of excised embryos is regarded as a more reliable & more exact test than the commonly used staining method for seed viability.

• Propagation of rare plant: As an abnormality some coconuts developed soft fatty tissue in place of the liquid endosperm. Such nuts are called “Makapum”. Being rare makapuros are very expensive and served only special banquets in the Philippines. Using the technique of embryo culture, De Gezman & Del Rosario succeeded in raising plant from makapuro nuts

• Clonal micro propagation: Embryos have high potential for regeneration & may be used for in vitro clonal propagation.

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