This article is reproduced here from The American Hosta Society
Bulletin # 11 1979-1980
Chloroplast Mutants in Hosta
The recent remarkable demonstrations of the mapping of plastogenes in Chlamydomonas (Gilham, 1974; Sager and Ramanis, 1973; 1976) should stimulate activity on research techniques that would be applicable to the analysis of the higher plant chloroplast genome (Wong-Staal and Wildman, 1973; Bedbrook and Bogorad, 1977). Unfortunately, there are few species among higher plants which offer the variety of unusual plastogene mutants needed for studies such as mapping (Tilney-Bassett, 1975). The Hosta, a genus of deciduous Oriental monocots, appears to be suitable for many kinds of chloroplast research because of the large number of mutants known of this type. However, the only extensive genetic work on any plastid system in Hosta was accomplished by Yasui in 1929. She concluded that all of the plastid mutations in variants of _H. sieboldii Ingram were maternally-inheriting mutants i.e. probably controlled by plastogenes. The present research reported here in extends Yasui's analysis to a larger number of species and varieties, notes some exceptions, and expands the interpretation of chimeral associations found in these plants.
Since many of the plastid mutants studied in this paper are maintained in chimeral condition with normal tissue, a brief discussion of plant chimera genetics is necessary to explain the interpretations of some of the mutant types (Neilson-Jones, 1969).
Through studies of chimeras in dicots, Dermen (1947, 1960) established the existence of three distinct layers of meristematic tissue which eventually determine the particular parts of the plant. Dermen's work indicated that, generally, in dicot leaves layer one (L-I) determines epidermal tissue, L-II determines the majority of subepidermal tissue, and L-III determines some internal tissue. As shown by Burk et al. (1964), ovules are determined primarily by L-II, with rare periclinal divisions resulting in replacement or displacement of L-II by either L-I or L-III (Stewart and Dennen, 1970). Thus, if a chimera with a plastogene mutant in L-II were self- or cross-pollinated virtually all of the progeny would be mutant. Therefore genetic analysis of self pollenated progeny of chimeras will tell us much of the histology of the mutation and will give some indication as to whether the character is under control of plastogenes or nuclear genes.
Materials and Methods
Plants used in the breeding experiments were obtained from several sources, as well as the author's own Athol, Ma. breeding program. Labeling of species follows the Flintoff (1972 and 1973) English translation of Maekawa's monograph on the genus. Names of hybrid clones are as established by the American Hosta Society registration committee, the official registration board for Hosta. Seedling numbers of clones are the author's own. Many different Hosta clones were cross- or self-pollenated and observed for the segregation of plastid types in the resulting seedlings. Blossoms were hand emasculated prior to crossing and the blossoms were protected from foreign pollination by reclosing the blossoms with rubber bands. Parental origin and stability of mutants among the seedling progenies were recorded.
The viable plastid mutants found in Hosta were classified in the following five classes: marginata, mediovariegata, aurea. Snow Flurry types, and mosaic [Chodat (1919) used the terms mediovariegata and marginata to describe Hosta clones and this terminology is retained].
Each of the classes is discussed separately because of the unique inheritance properties of each group. Results are summarized in Table 1 collectively with some of Yasui's data (1929) for a class from which the author was unable to obtain progeny.
1. Marginata (Fig. 1)
A Hosta clone with a marginata mutation has either a white leaf margin (albomarginata) or a yellow leaf margin (aureomarginata). When either class of marginata forms is used as a pod parent, regardless of the pollen parent, the progeny are virtually all green (Table 1). Among 293 seedlings from 12 progeny groups, there were 286 green, 4 variegated, and 3 yellow seedlings.
2. Mediovariegata (Fig. 2)
Mediovariegata mutants are distinguished from the other classes of plastid mutants by a large central area of each leaf visually devoid of green tissue. Albomediovariegata forms have white central areas of the leaf whereas aureomediovariegata have, for at least some times of the year,
yellow centers to the leaf. Although no progeny were grown by the author from albomediovariegata forms because of the considerable sterility encountered in some of these clones, Yasui obtained all white progeny from them. Progeny from aureomarginata clones used as pod parents were nearly all yellow regardless of the pollen parent (Table 1). Among 32 of the author's progeny (from 5 different matings) there were 2 greens, 2 variegated forms, and 28 yellows.
Aurea clones are, for at least some parts of the year, a chartreuse or yellow shade, distinct from the normal green color of most Hosta leaves. Seedlings from these clones when either self pollinated or when used as a pod parent in crosses are all aurea types. Three progenies containing 65 seedlings were observed (Table 1).
4. Snow Flurry mutants
Snow Flurry mutants are unique in that when they arise in spring they are nearly white (pale yellow-green) but gradually develop small green sectors throughout the leaves. Seedlings from self pollination of this type are all of this pattern but backcrosses to the heterozygous progenitor of these strains give a 1:1 segregation of normal green:Snow Flurry types.
5. Mosaic mutants
Clones in which segments of mutant tissue are interspersed between patches of normal tissue with no observable pattern are included in the mosaic group. Progeny from self-pollination of mosaic clones give a wide segregation of mutant and normal progeny depending at least somewhat upon the proportion of mutant tissue in the clone.
In numerous crosses using all green pod (?) parents, all of the progeny were green. Exceptions to this behavior are noted with the all green clone H. 'Snow Flakes' which gives rise to 25% variegated progeny when self pollinated and the all green clone H. 'Purple Profusion' which gave a progeny with some variegated offspring from self-pollination.
Stability of plastid types
Aside from the mosaic clones the other classes of mutants in Hosta are relatively stable. Occasionally shoots that are variegated will give rise to all green or all mutant shoots as in the series of mutants from H. 'North Hills' or H. 'Frances Williams' (Aden, personal communication), but these are rare. Clones generally reproduce their like from year to
year; e.g. generally marginata clones vegetatively produce only more of the same marginata clone.
One very unusual set of changes from one mutant clone to another is found in the H. undulata tribe (Fig. 2). H. undulata Bailey frequently undergoes spontaneous mutation to another form classified by Hylander (1954) as H. undulata Bailey var. univittata Hylander, a form with considerably less white tissue than the original _H. undulata. The ratio of the areas of green and white tissue change from 4:1 (white:green) in H. undulata to 1:5 (white:green) in H_. undulata var. univittata. Both of these forms give rise to an all green form known as H. undulata Bailey var. erromena Maekawa. The bloom scapes of these clones become increasingly tall as the percent of green tissue is increased in the leaf.
Mosaic clones may simply represent transient states before establishment of one of the mutant chimeral types or to all green or all mutant clones.
Besides this "long-term stability" of the various classes there is also a "season stability" of the clone. For example, one aurea clone may be a pale shade of yellow and slightly deepen in color as the season progresses whereas other aurea clones will emerge as a deeper yellow and gradually bleach to a paler shade. This tendency of either greening or bleaching seems to be inherited with the plastid trait but not enough cross data have been collected to ascertain precisely what the interaction is between nucleus and plastid in many cases (Nasyrov et al., 1975).
Some clones show virtually no change in pigmentation from spring to fall e.g. H. 'Frances Williams'.
Since the ovules are always formed by histogenic layer L-II (other than a few individuals arising from replacement or displacement), a plastogene mutant in this histogen will result in virtually all mutant progeny when this plant is selfed (Dennen, 1960). Thus, in Hosta, one may conclude that mediovariegata forms represent L-II mutations because virtually all mutant progeny are obtained from them. In contrast, in most dicots studied (Dennen, 1960; Burk et_ a_l., 1964), it was found that L-II mutations result in marginata forms.
The marginata mutants of dicots, when self pollinated, give rise to nearly all mutant progeny. In Hosta, marginata forms do not behave in this fashion; progeny from self pollination of marginata clones are virtually all non-mutant and therefore L-II is not the affected layer of marginata mutations in Hosta. Data of Dermen (1960) would indicate that it is a mutation in L-I that is responsible for the marginata phenotype in at least some monocots and it is likely that this is true for Hosta as well. Stewart (personal communication) feels that a fairly wide internal band of tissue in monocots is derived from L-I with L-II determining the remainder of the leaf. That marginata clones in Hosta should behave as L-I mutants is consistent with this notion.
In most dicots L-I produces only the epidermal layer so that mutations in this histogen are not recognized when they appear. By contrast the L-I of Hosta makes a sizable amount of submarginal tissue so that both L-I and L-II are clearly distinguishable and mutations may be recognized easily.
Mosaic clones with a strongly variegated central area to the leaf (L-II), when self pollenated, give a diverse segregation of plastid types. The non-Mendelian, completely independent segregation of plastid types suggests that the site of genetic control for most of these plastid mutants in the plastid itself (Tilney-Bassett, 1975). The large range of colors
and seasonal variation in Hosta plastids suggests that a complex genetic system within the plastid genome must exist to account for all these phenotypes.
Reciprocal crosses of both the author's (Table 1) and Yasui (1929) corroborate the analysis of the plastogene control of all of the plastid mutants excepting Snow Flurry mutants and a strong, if not full maternal inheritance (another possible exception is noted in Table 1).
An interested exception to the plastogene control of plastid inheritance is found in the Snow Flurry mutants. All of these clones stem from both the open and self pollination of H. 'Snow Flakes', a seedling of H. albomarginata Hylander var. alba Hylander. When H. 'Snow Flakes' is
self-pollinated a 3:1 ratio of normal green-.Snow Flurry types is found. When these variegated types are crossed back to H. 'Snow Flakes' either as a pod (5) or pollen (0 parent there is a 1:1 segregation of normal green:
Snow Flurry types. When self pollinated. Snow Flurry types give rise to more Snow Flurry types. All of these data would indicate that this mutant behaves as a simple Mendelian recessive. Since the only named clone of this type is H. 'Snow Flurry' the author has retained this name for thistype of plastid mutant and has labelled the gene controlling this type sf. Chlorophyll patterns present in the leaves of Snow Flurry types are reminiscent of anthocyanin patterns found in the Ds^Ac. and Snm controlling element systems of maize (McClintock, 1965; 1968). Kirk and Tilney-Bassett (1967) examined many types of similar cases and postulated the presence of a controlling element or controlling-element-like system. However, unlike the majority of mutants discussed by Kirk and Tilney-Bassett, Snow Flurry types have not given rise to any green progeny. This indicates that change of the repressed state (white), when a controlling agent is incorporated, to a normal state (green), when a controlling element is excised, does not occur when or where germ cells are being formed if,
indeed, the Snow Flurry mutant is a controlling element type.
The high percentage of mosaic variegated offspring from H. 'Purple Profusion* seemingly could not be explained as a controlling element system mainly because the seedlings behave like plants with sorting out variegation i.e. due to plastogenes (Kirk and Tilney-Bassett, 1967) rather than a controlling element system. All the variegated offspring displayed a mosaic phenotype initially ,characteristic of sorting-out variegation. Some of the shoots from these mosaic individuals gave rise to stable L-I (albomarginata) chimeras whereas others (e.g. Vaughn PP-1) are still in the sorting out stage. In PP-1 it is likely that the mutation is a plastogene mutant because of the cross data (Table 1).
Two explanations for the large number of mutant types from H. 'Purple Profusion' are possible: (1) H. 'Purple Profusion' may appear phenotypically green yet harbor small numbers of mutant plastids and the mosaic seedlings arise from sorting-out of these mutant plastids or (2) the plastids of these individuals inherit a susceptibility to environmentally-induced plastid mutation, possibly because of low levels of a repair enzyme for plastid DNA. We are now investigating at least the first of these possibilities.
An unusual spiral torsion mutation (de Vries, 1901) of a normal H. 'Frances Williams' was discovered in the author's plantings; this mutation results in varying degrees of "licorice stick" contortions to the bloomscape. When progeny are raised from the varying degrees of spiral torsioned clones, an increase in the number of replacements (from L-I) is observed, varying directly with the extent of the spiraling. This data may be an example of the simple physical placement mechanism that seems to be involved in replacement and displacement phenomena (Stewart and Dermen, 1970).
Insufficient progeny have been raised to blooming size to determine how the spiral torsion trait in Hosta is controlled as 3-4 years are needed
to reach blooming size. The changes in the H. undulata tribe also contain some interesting phenomena that deserve further comment (Fig. 2). In the change from H. undulata to H. undulata var. univittata, a shift in the green tissue derived from L-I has occurred that is not attributable to full replacement of L-II by L-I. Stewart and Dermen (1970) observe similar kinds of shifts of the proportions of green and white tissue in mericlinal chimeras and attribute this change to a shift in the number of apical initials which are included in the shoot apex. Perhaps similar changes in the relative amount of cells from one particular layer in a particular shoot apex or initial will result in changes like that found in the "mutation" of H. undulata to H. undulata var. univittata (Fig. 2). When either of the variegated forms revert to the all green H. undulata var. erromena a simple replacement of L-II by L-I best explains this situation, as excessive periclinal divisions of L-I may essentially eliminate the contributions due to L-II in a particular initial. It should be noted, however, that in the change from H. undulata to H. undulata var. univittata it is possible that a mutation of nuclear material may favor a larger contribution of L-I, resulting in _H. undulata var. univittata. Although Kanazawa and Akemine (1975) report abberant chromosome behavior in H. undulata both in pollen mother cells and to lesser extent in root tips, the all green var. erromena also exhibits this behavior. Because both green and variegated clones exhibit chromosomal abnormalities there may be no correlation between variegation changes and chromosomal changes.
Woods and DuBuy (1946) and Yasui (1929) noted seasonal "greening up" of the mutant tissue areas similar to that observed by the author in some of his material. This greening up differs from the usual behavior of plastogene mutants as most of the plastogene mutants studied in other genera start the season as normal-appearing green leaves and bleach to a
white or yellow color on exposure to light (von Wettstein, 1959). Although some of the plastid mutants (e.g. JH. 'August Moon') behave as those described by von Wettstein, other mutants show a greening behavior suggesting a lag period in the appearance of pigments similar to virescent mutants (von Wettstein, 1959; Kirk and Tilney-Bassett, 1967).
From the results reported here in Hosta appear to be uniquely-suited organisms for the study of chloroplast genetics. Some of their attributes are:
(1) the large number of different mutants available, includingboth nuclear (controlling element) and plastogene mutants;
(2) the ready identification of mutants as they appear becauseonly two histogenic layers make up the leaf and these layers are visually distinct;
(3) the mutations are not limited to a single species but occur in many of the species and varieties;
(4) several kinds of unusual changes in morphology or shifts in histogenic layers occur, some of which may be plastogene controlled.
In a study of plastid mutants of many species and varieties of the genus Hosta (Liliaceae) it was found that the majority of mutants were maternally inherited i.e. plastogene mutants, corroborating an analysis of Yasui (1929). The data from self and cross pollination of chimeral Hosta plant indicate L-I determines the epidermal and marginal subepidermal tissue with L-II determining the remainder of the leaf in these plants. Because of this unique histological situation plastogene mutants may be easily scored for as they occur.
In the Snow Flurry mutants a Mendelian recessive, and probably a controlling element, was shown to control the pattern.
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