Regularities of variability, constancy and instability of the genome, being the basis of formation and speciation, have not been investigated to a full degree yet. The role of cytoplasm in these processes has been examined still less. As a rule, the methods of reciprocal or backcrosses are used for investigation of a nuclear-plasm interaction. In these cases the cytoplasm of hybrid derivatives combines with the nucleus, in which the nuclear genomes of both parents are present completely or partially. Each of them can influence phenotypical manifestation of genes. The method of androgenesis in vivo allows exclusion in offspring of the nuclear genome of the maternal parent. The present paper reports the results of investigation of phenotypic manifestation of nucleus-cytoplasm interaction in alloplasmic androgenic maize lines on plant colour.
As androgen-inducing lines, WF9-T, WF9-S, and W23, inducing ability of which is conditioned by the gene ig, as well as the line AT-T, whose inducing ability has another nature, and the line HPL-1, were used. Line HPL-1 was produced from matroclinous haploid Kinelsky 103, and so a cytoplasm of the line HPL-1 was first marked as K-103 (Zavalishina and Tyrnov, MNL 69:120-121). Three inbred lines were used as nuclear donors: brown marker (BM), brown marker-96 (BM-96) and brown marker Saratovsky (BMS). All three lines have nuclear genes a B Pl R, conditioning brown colour of plants. The plants of lines BM, BM-96, BMS are very uniform in color and in quantitative signs. The inheritance of these signs was determined as a result of many years of observation. Line BM-96 was examined for 5 years, BM and BMS for about 20 years.
First, we produced androgenic haploids with the nucleus of one line (nuclear donor) and the cytoplasm of another (androgen-inducing). Haploids, as a rule, are mini-copies of the nuclear donor plants and preserve all their signs, including typical brown colour. However, on WF9-cytoplasm sometimes BM androgenic haploids were formed with changed colour - light-brown and almost green. Haploids were partially female fertile and, being pollinated by the line-nuclear donor, gave from 1 to 15 and more normal kernels with diploid embryos. The progeny of one haploid is an alloplasmic androgenic line, differing from line-donor by cytoplasm. Such a line, if it is characterized by CMS, can be constantly preserved in pure condition when pollinated by the line-nuclear donor.
Androgenic diploids were produced spontaneously on cytoplasm of HPL-1: there were 2n-2n twins with BM nuclear genome and also monoembryonic androgenic diploids with nuclear genome BM-96. Phenotypically these diploid androgens conformed completely to the nuclear donor plants. After self-pollination they also gave pure lines.
As a result, alloplasmic lines with nucleus BM were produced on cytoplasms WF9-T, WF9-S, W23, AT-T; with nucleus BMS on cytoplasms W23, AT-T, HPL-1; with nucleus BM-96 on cytoplasm HPL-1.
Now, we apply this to analysis of plant colour inheritance.
All androgenic haploids and their progenies were crossed to nuclear line donors. By this, in progenies of androgenic haploids, segregation of plant colour took place. In addition to typical brown plants, light-brown, sunlight brown (tun) and green plants were discovered. Distibution of plants by colour was rather relative, since among light-brown plants there were as many intensely coloured ones, as less. Analogously, among sun light brown (tun) plants colour was more manifest in some, and only weak traces of brown colour on the husks and tassel glumes in others. There were not even traces of brown colour in green plants. Sometimes there were plants which can be related to any group only with difficulty: for example, a few plants had light-brown tassels, but green culms, husks, sheaths, and in one plant all organs were brown, but culm, hidden in sheaths, was green.
Change of plant colour is accompanied by change of cob colour. The cobs of light brown plants were fawn-coloured, and cobs of sun-light brown and green plants were white.
Segregation of plant colour in progeny of typical androgenic brown plants appeared by crossing them to brown plant nuclear donors. For example: androgenic haploid BMS with cytoplasm AT-T had brown colour. By crossing it to nuclear donor 6 kernels were set. Four brown and 2 light brown plants were grown from them. In the progeny of one brown plant, when it was crossed to brown nuclear donor, 13 of 60 plants were brown, 21 were light brown, 12 were sun-light brown (tun) and 14 were green. By crossing light brown androgenic plants to brown nuclear donor, in progeny there were light brown, sun-light brown and green plants. Brown plants were not discovered at all.
For example: light brown x brown gave 4 light brown, 22 sun-light brown, and 60 green. From sun-light brown by the nuclear donor, only sun-light brown and green plants were formed, and sometimes only green ones.
For example: sun-light brown x brown gave 1 sun-light brown and 26 green.
In all these crosses quantitative correlation of the plants differing by colour was different. And progeny of green androgenic plants, when they were pollinated by brown nuclear donor, during a number of generations only green plants appeared, which sometimes became dark green.
Analogous segregation was observed in progeny of alloplasmic lines with fertile cytoplasm by self-pollinating. Self-pollination of androgenic green plants led to only green plants.
In addition, progenies from reciprocal crosses of androgenic green plants to brown plants of nuclear donor were analyzed. In F1 only green plants appeared. The same was observed in F2 of these crosses. When androgenic diploids BMS on cytoplasm HPL-1 were self-pollinated, in progenies (in both members of twins) deviation in manifestation of plant colour was not discovered during 15 generations: all plants were brown. In self-pollinated progeny of BM-96 on the same HPL-1 cytoplasm half of the plants had a typical brown colour, and another half were green. Green plant colour was preserved by self-pollination in following generations. The progenies of the brown plants were not investigated.
It has to be noted that colour changes in progeny of androgenic plants happened in the same direction (from brown to green), but manifested in a different manner. Sometimes they manifested slowly during a number of generations, which looks like the result of accumulation of gene-modifiers. In other cases changes happened unevenly, and green colour manifested already in the next generation, which is analogous to mutation. Earlier we tried by the backcross method to transfer the inbred line BM on cytoplasms of lines with S-, T- and C- types of CMS. After two-three backcrosses uniform progeny were produced, consisting of brown plants only. However, following further backcrosses plants with light brown and green colour appeared. In spite of our attempts to select as maternal parents the most brown plants, only green plants appeared in progeny.
All the foregoing speaks about non-casual character of brown colour changes in the plants of alloplasmic lines. It is known that in maize a number of series of multiple alleles take part in determination of plant colour (Emerson, Cornell. Univ. Agr. Exp. Sta. 39:1-156, 1921; Coe and Neuffer, In Sprague (ed): Corn and Corn Improvement: 111-223, 1977; Coe, The Maize Handbook: 279-281, 199).
We made an attempt to control the presence of recessive alleles of genes B and Pl in plants with changed colour by the method of analysing crosses. We used a line having the genes A b pl R-nj Cudu. BM and BMS plants (nuclear donors) were crossed to this line. In F1 all plants had intense dark-purple colour. That colour corresponded to genotype A B Pl R.
Androgenic plants with brown, light brown, sun-light brown and green colour were also pollinated by the line with genes A b pl R-nj Cudu. In F1 of these combinations we observed, correspondingly, purple, light-purple, sun-purple, sun-light purple plants.
In addition to the described plants in F1 plants with other colours appeared: a) by pollinating androgenic BMS plants on cytoplasm AT-T purple plants appeared, the leaves of which at the top of husks were green; b) by pollinating brown plants with green stem purple plants appeared with free designs on purple colour of husks, sheaths, tassels, and culm inside sheaths was green; c) by pollinating green plants the most various plant colour progenies arose: sun-dilute purple, sun-red purple, sun dilute purple with dark anthocyanic spots on culm or with purple stripes along culm, as well as lilac-pink and, at last, plants with very weak colour, almost green; d) by pollinating androgenic BMS on cytoplasm HPL-1 purple plants arose, the culms of which inside of sheaths had granite colour.
From all the foregoing it follows that transfer of dominant genes B and Pl into another cytoplasm provokes a change of plant colour, that phenotypically looks like the result of the presence of a series of multiple alleles and the recessive gene pl. The character of segregation, differing from the Mendelian one, is analogous to that described earlier in maize by other authors (Coe, Genetics 53:1035-63, 1966; Brink, Ann. Rev. Genet. 7:129-152, 1973) and was attributed to paramutations, the cause of the origin of which is not known yet.
Since in our experiment we used pure material, and the method of androgenesis in vivo allowed transfer of the same genome into the different cytoplasms, the single origin of paramutagenicity could be cytoplasm. We can suggest a scheme for the foregoing process. Under the influence of new cytoplasm the dominant gene becomes paramutable, transferring to the condition of one of its recessive alleles and acquiring by that the character of paramutagenicity. Under its action allele B, being brought by the nuclear donor, acquires the same characters.
Since the effect of paramutagenicity was observed, practically, in all combinations, excluding BMS on cytoplasm HPL-1, we can speak about the universality of this manifestation of nucleus-cytoplasm interaction.
Does a gene return to the previous functional condition by transfer to the initial cytoplasm?
To check this, we crossed plants of line-nuclear donor to androgenic green plants. All F1 plants had green colour. Then we again pollinated plants of the nuclear donor by F1 pollen. In the progeny of this cross all plants were green. After the second backcross among 50 plants two light brown plants with brown tassels were discovered.
This speaks of the possibility of restitution of functional activity of nuclear genes. By that the process of restitution proceeds gradually like its change under the influence of new cytoplasm. Thus, changes of genes as a result of paramutations are rather stable, and genes preserve phenotypical effect even being returned into their own cytoplasm. And only with the course of time do they return to their initial condition. In our report we considered only changes of colour manifestation. It can not be excluded that other genes, determining the quantitative traits, are under the same or analogous dependence on cytoplasmic factors. Coming from this suggestion, any cross can give new combinations of nuclear genome and cytoplasm, and their interaction can be manifested in the following generation in the form of spontaneous mutations, gene-modifiers, paramutations, mobile genetical elements, etc.
It can be supposed, that it is a cause of significant variability of
characters and, in the end, a cause of line variation and aging. In all
probability, this has to be taken into account in production of new sorts,
since formation of some characters can not proceed according to simple
schemes for ordinary nuclear or cytoplasmic inheritance.
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