Embryo development and embryo culture

Development of Embryo (Embryogeny):-

> As written earlier also, the highly organized body of a seed plant represents the sporophytic phase of the life-cycle. It begins its existence usually with the fertilized egg, the zygote, which develops into the embryo by characteristic steps showing characteristics of future adult organization of the plant. Embryo, as a whole, assumes a specific form in which an axis and one or more leaf like appendages, the cotyledons, can be recognised.

> Because of its location below the cotyledons, the stem like axis is called hypocotyl. At its lower end (i.e., the root pole), the hypoectyl bears the incipient root, at its upper end (i.e., the shoot pole), above the caryledons, the incepient shoot. The root may be represented by its meristem (apical meristem of the root) or by a primordial root, the radicle. Similarly, the apical meristem of the shoot located at the shoot pole may or may not initiate the development of a shoot above the cotyledons. If a primordial shoot is present, it is called epicotyl or plumule.

1. Development of Embryo in Monocot Plants:-

> In monocotyledonous plants, a good deal of variation is found in the stages of development of embryo. It is rather difficult to find a single plant in which the development of embryo may be considered as typical of monocotyledons. Development of embryo in Sagittaria (family, Allismaceae) has close resemblance to that in other monocots and is thus described here.

> Soon after fertilization, the fertilized egg or oospore greatly enlarges in size and divides transversely to form a 3-celled proembryo. These three cells are basal, middle and terminal. The basal cell, which is cut off towards the micropylar end, enlarges very much and forms major portion of the suspensor. The middle cell undergoes repeated transverse and vertical divisions thus differentiating few suspensor cells, radicle, plumule and hypocotyl. The terminal cell also undergoes a number of divisions in various planes and forms a single cotyledon.

> Here the cotyledon is a terminal structure and the plumule is laterally situated in a depression. In some monocots, like Colocasia, there is no suspensor in the embryo. Suspensor usually pushes the embryo into the endosperm from where the former gets nourishment. The suspensor may also serve as haustorium to absorb food from the nucelius. In some monocotyledonous plants like Agapanthus (family, Liliaceae) two cotyledons are formed instead of one.

2. Development of Embryo in Dicot Plants:-

> Development of embryo in dicotyledonous plant basically follows a uniform pattern except for slight variations. The fertilized egg or oospore greatly enlarges in size, elongates and divides transversely into a suspensor cell (towards the micropylar end) and an embryonal cell (towards the middle of the embryo sac). As the development proceeds, the suspensor cell further divides and forms a 8 to 10-celled suspensor which pushes the developing embryo into the food storage tissue, endosperm. The lowermost cell of the suspensor, which is called hypophysis, undergoes more divisions to form the radicle.

> These changes in the suspensor cell are associated with the enlargement of the embrynonal cell. The embryonal cell also divides; the first division is longitudinal followed by one more longitudinal at right angle to the first and the other transverse forming an octant (8-celled proembryo). This is followed by a periclinal division thus forming a 16-celled structure which may be differentiated into posterior or hypobasal octant (towards the suspensor) and anterior or epibasal octant. The epibasail cells, as a result of further divisions and differentiation, form plumule and cotyledons, while the hypocotyl is formed from the hypobasal cells.

> The peripheral cells of the proembryo subsequently divide by anticlinal walls, thus differentiating a single-layered dermatogen (it forms epidermis). The middle-cells of the proembryo also divide periclinally and form plerome (which forms vascular tissue). The tissue in between dermatogen and plerome is known as periblem (it forms the cortex).

> As the development further proceeds, the free end of the developing embryo becomes heart-shaped, The two lobes of the ‘heart’ are the primordia of the cotyledons. Plumule is situated in the depression in between two cotyledons. Unlike monocot embryo, here the cotyledons are lateral and the plumule is terminal.

> As in Capsella bursa pastoris, the embryo enlarges rapidly consuming the surrounding endosperm. Thus the seed so formed becomes non-endospermic. The endosperm may not be completely exhausted and persists in the seed; such condition is found in endospermic seeds leg:, castor).

भ्रूण संवर्धन (Embryo Culture):-

·         परिभाषा (Definition):- परिवर्धित हो रहे बीज में से तरुण भ्रूण को निकालकर निर्जमित दशाओं में पोष पदार्थ पर संवर्धित करने की प्रक्रिया को भ्रूण संवर्धन कहते हैं।

(Embryo culture is a process in which young embryo removed from the developing seed and cultured on artificial medium in sterilized conditions.)

·         सिद्धान्त (Principle):-

Ø  सामान्यतया पोष पदार्थ पर तरुण भ्रूण का परिवर्धन या विकास पूर्ण नहीं हो पाता है और यह सीधे ही पौधे में विकसित हो जाता है। पोष पदार्थ में निम्न में से कोई एक मिलाने से भ्रूण का समय से पहले अंकुरण रोका जा सकता है –

(Generally, the growth or development of the young embryo on the artificial is not complete and it develops directly into the plant. Premature germination of the embryo can be prevented by adding one of the following to the artificial medium-)

i. उच्च सुक्रोज सांद्रता (High sucrose concentration)

ii. उच्च ABA सांद्रता (High ABA concentration)

Ø  कुछ पौधों में परिपक्व बीजों के भ्रूणो का भी संवर्धन किया जाता है जैसे – Iris, Orchids आदि।

(In some plants, embryos of mature seeds are also cultured such as - Iris, Orchids etc.)

Ø  ग्लोबुलर अवस्था के बाद के भ्रूणो का कृत्रिम संवर्धन अधिक आसान होता है। परन्तु क्रूसिफेरी कुल के कुछ पौधों में केवल कुछ कोशिकाओं वाले भ्रूणो का संवर्धन करना संभव हो सका है।

(Artificial culturing of embryos after the globular stage is easier. But in some plants of the Cruciferae family, it has been possible to culture embryos with only a few cells.)

Ø  तरुण भ्रूण को भृणपोष पर संवर्धित कर सकते हैं ताकि इसका पूर्ण विकास व परिवर्धन हो सके।

(Young embryo can be cultured on the endosperm so that it can be fully developed and grown.)

·         विधि (Procedure):-

Ø  फल से अपरिपक्व बीजों को पृथक करके 10 मिनट के लिए 5℅ Teepol विलयन (एक द्रव डिटरजेंट) में डुबोकर रखते हैं। अब Teepol को हटाने के लिए कर्तोतकों को पानी से धो लेते हैं।

[Separate immature seeds from the fruit and keep them immersed in 5℅ Teepol solution (a liquid detergent) for 10 minutes. Now wash the explants with normal water to remove Teepol from the surface.]

Ø  अब इन अपरिपक्व बीजों के सतही निर्जमीकरण के लिए 60 सेकंड के लिए 70℅ Ethanol में डुबोकर रखते हैं। 5 – 10 मिनट के लिए 0.1℅ HgCl2 के विलयन में डुबोकर रखते हैं। अतिरिक्त रसायन को सतह से हटाने के लिए 3 – 4 बार निर्जमित आसुत जल से धो लेते हैं।

(Now for surface sterilization of these immature seeds, immerse them in 70℅ Ethanol for 60 seconds. Then immersed in 0.1℅ HgCl2 solution for 5 - 10 minutes. To remove excess chemical from the surface, wash it 3-4 times with sterilized distilled water.)

Ø  अब अपरिपक्व बीज पर चाकू या ब्लेड की सहायता से कट लगाकर सुई की सहायता से तरुण भ्रूण को पृथक कर लेते हैं। जिसे अब कर्तोतक कहा जाता है।

(Now the immature seed is cut with the help of a knife or blade and the young embryos are isolated with the help of a needle. Which is now called explant.)

Ø  अब इन पृथक किए गए भ्रूणो को माइक्रो पिपेट या छोटे चमचनुमा स्पैचूला की सहायता से 10 cm पेट्रीडिश में 25 ml ठोस अगार माध्यम पर स्थापित करते हैं। सामान्यतया 6 – 8 भ्रूणो का संवर्धन एक पेट्रीडिश में किया जा सकता है। माध्यम में सुक्रोज या ABA की सांद्रता उच्च रखी जाती है। पेट्रीडिश को सैलो टेप से सील बन्द कर देते हैं ताकि माध्यम के शुष्कन को रोका जा सके।

(Now these isolated embryos are placed on 25 ml solid agar medium in 10 cm patridish with the help of micro pipette or small spoony spatula. Normally 6 - 8 embryos can be cultured in a patridish. The concentration of sucrose or ABA in the medium is kept high. Patridish is sealed with cello tape to prevent drying of the medium.)

Ø  अब इस पेट्रीडिश को इंक्यूबेटर में रख देते हैं जहां इसे 25 ±1°C तापमान व 16 घण्टे का प्रकाशकाल दिया जाता है। प्रकाश देने के लिए ठंडी सफ़ेद फ्लोरीसेंट ट्यूब का उपयोग किया जाता है।

(Now put this patridish into an incubator where it is given a temperature of 25 ± 1 ° C and 16 hours of light period. Cold white fluorescent tubes are used in incubator to provide light.)

Ø  लगभग 4 सप्ताहों में भ्रूण विकसित हो जाता है और प्ररोह तंत्र व मूल तंत्र बनाना शुरू कर देता है।

(In about 4 weeks, the embryo develops and starts forming the shoot system and root system.)

 Øअब इसे नए ताजा माध्यम पर बड़ी संवर्धन ट्यूब में स्थापित करके उप – संवर्धन कर लेते हैं।जिससे नया पौधा विकसित हो जाता है।

(Now sub-culture it by establishing in a large culture tube on a new fresh medium, so that a new plant develops.)

·         अनुप्रयोग (Applications):-

i. Embryo Rescue (भ्रूण बचाव):- दूरस्थ संकरण से बना भ्रूण भ्रूणपोष के ह्रास के कारण नष्ट हो जाता है। इसे नष्ट होने से बचाने के लिए तरुण बीजों से भ्रूण को निकालकर कृत्रिम रूप से माध्यम पर संवर्धित करते हैं।

(Embryos formed by distant hybridization are destroyed due to degeneration of endosperm. To protect it from destruction, extract the young embryo from the seeds and artificially grows it on the medium.)

उदाहरण (Example):- 

जौं X राई (Barley X Rye)

 जौं X गेहूँ (Barley X Wheat)

गेहूँ X राई (Wheat X Rye) = Triticale या Man made Wheat का निर्माण

[Development of Triticale or Man made Wheat]

ii. अगुणित पादप उत्पादन:- अंतराजातीय संकरण में जब जाइगोट से भ्रूण विकसित हो रहा होता है तो एक जाति के गुणसूत्र नष्ट हो जाते हैं तथा भ्रूण अगुणित हो जाता है। इस अगुणित भ्रूण के कृत्रिम संवर्धन से अगुणित पौधे प्राप्त किए जाते हैं।

(In inter-specific hybridization, when the embryo is developing from a zygote, the chromosomes of one species are destroyed and the embryo becomes haploid. The haploid plants are obtained by artificial culturing of this haploid embryo.)

उदाहरण (Example)– Hordeum bulbusum  X  जौं या गेहूँ  (Barley or Wheat)

(इस क्रॉस में Hordeum bulbusum के गुणसूत्र विलुप्त हो जाते हैं।)

(In this cross the chromosomes of the Hordeum bulbusum become extinct.)

iii. Orchid प्रवर्धन:- Orchid के बीजों में भ्रूण तो होता है परन्तु भ्रूणपोष का अभाव होता है। अत: इनके कृत्रिम संवर्धन से पौधे आसानी से विकसित किए जा सकते हैं।

(Orchid seeds contain embryos but lack endosperm. Therefore, plants can be developed easily with their artificial culture.)