SK281132B6 - Method of induction of creation of somatic embryos - Google Patents

Abstract

Somatic embryos are prepared from undifferentiated plant cells which prior to forming new structures are first cultivated in an induction medium with the content of growth material (benzylaminopurine, yeast extract, glutamine saccharosa, dicamba, 2,4-D). The new structures thus formed are then cultivated in a basic cultivation medium with the content of saccharosa until a phase in which the somatic embryo is capable of sprouting is reached.

Description

Technical field

The invention relates to the induction of somatic embryo formation, providing new individuals with the same properties as the original plant.

BACKGROUND OF THE INVENTION

Despite advances in plant biotechnology and genetic engineering research, the possibility of multiplying plants with the desired targeted properties remains a serious problem. Plants propagate (multiply) in in vitro cultures after transformations and mutations. Three micro-propagation methods can be used:

direct and indirect organogenesis (separate induction of roots and shoots); direct and indirect embryogenesis (induction of bipolar, embryo-like structures) and propagation through existing meristems.

The success of any micro-propagation method depends on the genotype of the plant species concerned, the composition of the nutrient medium as well as the culture conditions (Biotechnology in Aericulture and Forestry 3, "Potato" ed. YPS Bajaj, 1987, p. 509. Springer Verlag, Berlin, Heidelberg, New York, London, Paris, Tokyo (1988). From the economic point of view, the most effective multiplication is through the formation of somatic embryos (the embryo is derived from plant body cells, not gametes).

Increasing the resistance of the plant species (eg potatoes) is one of the most important aims.

ground seeds ". A further advantage lies in the speed of said process - from one explanter, up to 20 somatic embryos can be obtained in 30 to 40 days, with a multiplication coefficient of 3. 10 ² to 3. 10 ⁴ .

SUMMARY OF THE INVENTION

We have now found the possibility of inducing somatic embryo formation by the method of the invention. It is the object of the invention that undifferentiated plant cells are first cultured on an induction culture medium containing growth substances until new structures (e.g. callus) are formed. The newly formed structures are then grown on a basic culture medium containing sucrose until the stage of the somatic embryo able to germinate (normally developed root, hypocotyl, germinate leaves). It is possible to cultivate undifferentiated cells first in an induction culture medium containing 0.03 to 0.5 mg of benzylaminopurine, 0.1 to 0.6 mg of glutamine, 1 g / ^{l of} yeast extract as well as 2 % to 10 wt. of sucrose for 20 to 30 days, then the newly formed structures are separated from the explants and grown on a sucrose-containing basic culture medium until the somatic embryo germinable stage. The second possibility is the first of the undifferentiated plant cells are cultured on the induction culture medium containing 1.0 to 3.0 mg · l ¹ dicamba or 2,4-D in the callus, which is repeatedly passaged until the establishment granular embryogenic callus . The granular embryogenic callus is then grown on a basic culture medium containing sucrose up to the stage of the somatic embryo capable of germination. As the basic culture media, so-called " B ₅ media (according to Gamborg), Murashige-Skoog media and the like, which are usually agar-reinforced.

An advantage of the method of inducing the formation of somatic embryos according to the invention is that a large number of new plants with the same characteristics as the parent plant (genetic uniformity of new plants) can be obtained from one immature embryo. After coating with alginates, so-called embryos can be prepared from somatic embryos. Examples of the invention

Example 1

Immature seed-derived zygote embryos were extirpated in the late heart to torpedo stage and cultured in a Petri dish on Gambor's induction culture medium (B ₅ medium) containing 0.05 mg.l ¹ benzylaminopurine (BAP), 1. ¹ g · l yeast extract, 0.4 mg · l ¹ glutamine and 5% by weight. sucrose. Cultivation conditions: 25 ° C (± 2 ° C), 16/8 light / dark light mode, illuminance 3000 - 3500 lux.

New structures were formed from the original explants epidermal cells within 20 days and transferred to a Petri dish containing 6% w / w basic culture medium. sucrose and cultured for 10 days. After this time began to form so-called. somatic embryos with normally developed root hypocotyl and cotyledons, able to germinate immediately.

During 40 days, 20 somatic embryos were obtained from one explante.

Example 2

Sterile immature zygote embryos are cultured in a Petri dish on an induction culture medium according to Murashige-Skoog (1962) containing 2 mg.l ^{-1 of} 12,4-dichlorophenoxyacetic acid (2,4-D). Within 1-14 days, disintegrating white callus was formed, which was repeatedly passaged 4 times on fresh induction culture medium every 14 days, thereby intensifying cell division in callus and activating the embryogenic potential of each tolipotent plant cell. After these 4 passages, granular embryogenic callus was formed and transferred to the basic culture medium containing 4 wt. sucrose, where embryogenic cells and new plants developed. From 300 to 3000 embryos were obtained per explant.

Example 3

The procedure was as in Example 2 except that dicamba (3,6-dichloro-2-methoxybenzoic acid) was added to the induction culture medium in an amount of 3 mg.l ^-1 and sterilized potato leaf pieces were used as undifferentiated plant cells. After callus induction, this was passaged 3 times every 2 weeks to fresh sucrose culture medium.

Industrial usability

The invention is applicable to all biotechnological processes such as transformation (gene transfer enhancing utility, disease resistance), mutation and the like.

Claims (5)

PATENT CLAIMS A method for inducing the formation of somatic embryos, characterized in that undifferentiated plant cells are first cultured on an induction culture medium containing growth substances until the formation of new structures which are then grown on a basic culture 2 Sucrose-containing storage medium until the germ stage of the somatic embryo is able to germinate. The induction method according to claim 1, characterized in that the undifferentiated plant cells are first cultured in an induction culture medium containing benzylaminopurine, yeast extract, glutamine and sucrose for 20 to 30 days, the newly formed structures are separated from the explante and grown on a baseline. sucrose-containing culture medium. The induction method according to claim 2, characterized in that the induction culture medium contains 0.03 to 0.5 mg benzylaminopurine, 0.1 to 0.6 mg glutamine, 2 to 10 wt. sucrose and 1 g / ^{l of} yeast extract. The induction method according to claim 1, characterized in that undifferentiated plant cells are cultured on an induction culture medium containing dicamba or 2,4-D, the resulting callus is repeatedly passaged to form granular embryogenic callus, which is then grown on a basic culture medium. sucrose-containing medium to the somatic embryo stage capable of germination. The method of claim 4, wherein the induction culture medium contains 1 to 3 mg.l -1 of dicamba or 2,4-D.