Biotechnology of obtaining
in vitro
of spring soft wheat hybrids
with fixed heterosis effect
Prof. N.N.Kruglova, Institute of Biology of Ufa Sci. Centre of Russian Academy of Sciences, Ufa.
Prof. T.B.Batygina, Komarov Botanical Institute of Russian Academy of Sciences, St.-Petersburg.
The biotechnology of stable reception and duplicating of spring soft wheat androcli-nalhybrid plants with fixed heterosis effect is developed. The given biotechnology is based on use of a cell-engineering method of isolated anthers culture in vitro with attraction the data of plant embryology. Competitive advantages of the developed biotechnology are analyzed.
Introduction
An anther is a fertile part of a stamen, in its holes the cell of the so-called sporogenous tissue form microsporocytes, which undergo a meiosis and give a rise to haploid microspores. Microspores grow in pollen grains – male gametophytes.
However, under strictly determined controlled conditions of in vitro culture, as a rule, under the influence of stress factors, it is possible to switch the program of development of that anther cell which gives rise to a male gametophyte – a pollen grain under natural conditions. In other words, in experimental system of in vitro culture such a cell at the definite stage of its development is capable of (morphogenetic competent) a change of development program from usual gametophyte to sporophyte one leading to formation of haploid plant-sporophyte. In this case plant reproduction passes by the scheme: sporophyte à sporophyte.
The phenomenon of sporophyte formation from morphogenetically competent anther cell (under the conduction of in vitro culture) was revealed in 1964 at the example of Datura L. [Guha, Maheshwari, 1964]. The discovery of this phenomenon may be considered one of the most significant in plant biology in our days. The process of sporophyte plant formation from morphogenenetically competent anther cell in culture in vitro was called androcliny (from Greek ανδρος - male, κλινος - in clined to). In western literature the term anrdogenesis in vitro is widely spread.
Thus, the core of the most interesting phenomenon – anrdrocliny (or anrdogenesis in vitro) – consists in changing of development program of anther morphogenetic competent haploid cell from habitual gametophyte way (pollen grain formation) to the other way – sporophyte (haploid plantlet formation).
Competitive advantages of biotechnology of reception of androclinal plants
The biological phenomenon of androcliny lies in the basis of the method of isolated anther in vitro culture – the unique biotechnology method which is perspective in modern genetic and selection investigations of plants. Uniqueness of this method is that in the present day it is the only method for fixation of valuable heterosis effect of 1-st generation hybrids.
Competitive advantages of this biotechnology consist in reception for rather short time the homozygous constant haploid hybrids of 1-st generation, keeping in a genotype economic valuable attributes of parental forms. Such hybrids are very important in the modern agricultural practice.
The using of haploid hybrids considerably facilitates also selection of valuable genotypes, arising in result of recombination of genetic data of parental forms. Such selection enables to speed up an estimation of perspectivity of the received hybrids.
Conversion of haploids into dihaploid status allows to receive high-grade seeds of such plants.
Features of the prepared biotechnology of reception of androclinal spring soft wheat plants
In the laboratory of plant genetics and cytology of the Institute of Biology of Ufa Sci. Centre of RAS (Ufa) in creative cooperation with the Laboratory of embryology and reproductive biology of Komarov Botanical Institute of RAS (St.-Petersburg) the laboratory sample of biotechnology of stable mass reception in culture in vitro of economic valuable and competitive dihaploid hybrid plants of wheat spring soft with fixed heterosis effect was prepared. Features of the given biotechnology consist in using of cell-engineering method of isolated anthers culture in vitro as well as the data of plant embryology, cytology, histology and physiology. All this conducts to maximum full realization of morphogenetic potential of androcliny initial cells in conditions of culture in vitro.
It was experimentally revealed that androcliny initial cells in spring soft wheat are microspores at the highly vacuolated phase (according to periodization by [Êðóãëîâà, 1999, 2001]). On our opinion, the ability to switching the program of development of highly vacuolated microspore from gametophyte to sporophyte one is determined by a number of moments. Firstly. The peculiarity of structural organization of this cell: the presence of wall central vacuole and a large nucleus located in opposition direction to a germ pore. Thus, a highly vacuolated microspore is characterized by well expressed polarity (apical-basal organization). It should be noted that by this attribute a highly vacuolated microspore is structurally similar to a mature egg cell of the majority of flowering plant. It confirms the existence of principal similarity in the organization of initial cells of new individuum at different systems of reproduction under natural conditions and in culture in vitro. Secondly. Active metabolism processes in highly vacuolated microspore, which are confirmed by the data of ultrastructural analysis. All these features characterize a highly vacuolated microspore as a an extremely active cell.
Phenotypic traits of spring wheat donor plants, containing anthers with highly vacuolated microspores are as follows: spike top in leaf cover is located at one fourth of distance from the base of flag leaf to the base of next to bottom leaf (the VII-th stage of organogenesis). Such phenotypic traits serve as the morphological markers for express diagnostics of donor plants and so for optimization of the process of haploid production.
As a result of experimental researches it was established that androcliny induction was promoted by stress cold influence in empirically revealed regime (+40Ñ, 7 days) at anthers before their inoculation into nutrient medium in vitro. What is the mechanisms of cold effect? It was experimentally established that cold stress provoked “break-off” of highly vacuolated microspores from anther wall. Such “break-off” leads to the break of anther integrity as a system, disturbance of morphogenetic correlations between anther wall tissues and highly vacuolated microspores and by that to infringement of determination of normal development of pollen grain. Besides, this separation leads to the change of microspore structural organization (change of its polarity) and to the changes of anther wall (its degeneration). Thus, a trigger of sporophyte way of highly vacuolated microspore morphogenesis in vitro is a cold stress effect in a definite regime.
After cold stress influence anthers inoculated into in vitro conditions on induction nutrient medium Potato II [Chuang, Ouyang, 1978] in own updating [Êðóãëîâà, Áàòûãèíà, 2002, 2006].
In conditions of culture in vitro the morphogenesis of highly vacuolated microspores undergo in different pathways. Naturally, biotechnologists are interested in the pathway of morphogenesis which leads to formation of high-grade fructifying plantlets. It is revealed that optimal way of morphogenesis in this case is embryoidogenesis consisting in the formation of embryoid (from Greek εμβρυον - a germ, ειδος - an image) – embryo-like structures (synonyms: a somatic embryo, a advential embryo).
It was established that induction of embryoid formation is defined by the balance between concentration of exogenic auxin 2,4-D in modified induction nutrient medium Potato II and the content of endogenic auxin IAA in anther at the moment of inoculation. For each wheat cultivar or line this the balance will be the individual. Selection of this balance as the methodical approach allows to manipulate the process of highly vacuolated microspore morphogenesis in culture in vitro and promotes acceleration of biotechnological production of spring wheat androclinal plantlets.
on the basis of detailed embryological, cytological and histological data and also the morphological observations the main developmental stages of initial highly vacuolated microspore were established up to the embryoid formed on induction nutrient medium Potato II. It was shown that microsporial embryoid development in in vitro condition was essentially similar to zygotic embryo development in natural conditions.
Formed embryoids transferred on the nutrient medium for the regeneration, which made according [Blaydes, 1966] in own updating [Êðóãëîâà, Áàòûãèíà, 2002, 2006]. Received androclinal plantlets raised in test tubes up to the phenophase of tillering. Further plantlets were taken from test tubes and by the help of cytogenetic control selected only haploid individuals. Them processed a mix for dihaploidization (composition of the mix is know how). After cytogenetic control the dihaploidized plants were transferred into soil where they developed to phenophase of full grain maturity.
The laboratory and field estimation has shown high germination power of received seeds of androclinal plants. Quality of seeds is confirmed with the data of embryological analysis.
Conclusion
Biotechnological method of obtaining of high-grade fertile androclinal dihaploid plants of spring soft wheat is the complicated process depending on the complex of various factors. However already now the arsenal of theoretical and experimental data allows to make this process controllable and to produce full value competitive androclinal plants with fixed heterosis effect for their introduction into selection practice. High-grade seeds allow to duplicate the received androclinal plants.
Scope of the developed biotechnology is the breeding of new zoned cultivars of spring soft wheat with economic valuable parameters.
The basic features of the given biotechnology are protected by a number of copyright certificates. Authors published the methodical recommendations on use of the biotechnology [Êðóãëîâà, Áàòûãèíà, 2002, 2006].
The laboratory sample of the biotechnology has passed successful approbation in field conditions of the Selection center of the Bashkir Scientific Research Institute of an Agriculture of Russian Academy of Agrarian Sciences (Ufa) at creative commonwealth with the laboratory of selection of a spring wheat of this Institute.
Researches are maintained by the RFBR (grants ¹ 99-04-48002, ¹ 99-04-48496, ¹ 00-15-97828, ¹ 01-04-06565, ¹ 02-04-48701, ¹ 02-04-49807, ¹ 02-04-06132, ¹ 03-04-06213, ¹ 05-04-97911, ¹ 05-04-08114), by the program «Integracia» (grants ßÎ 128/1644 è Ö004) and by program «Leading scientifis schools of Russian Federation» (grants ¹ ÍØ 2148.2003.4, ¹ ÍØ 4834.2006.4).