defective of maize. i. - home | genetics · 2003. 7. 29. · defective kernel mutants of maize. i....

DEFECTIVE KERNEL MUTANTS OF MAIZE. I. GENETIC AND LETHALITY STUDIES

M. G. NEUFFER

Department of Agronomy, University of Missouri, Columbia, MO. 65211

AND

WILLIAM F. SHERIDAN

Department of Biology, University of North Dakota, Grand Forks, N . D. 58202

Manuscript received July 9, 1979 Revised copy received April 21,1980

ABSTRACT

A planting of 3,919 MI kernels from normal ears crossed by EMS-treated pollen produced 3,461 MI plants and 3,172 selfed ears. These plants yielded 2,477 (72%) total heritable changes; the selfed ears yielded 2,457 (78%) recessive mutants, including 855 (27%) recessive kernel mutants and 8 (0.23%) viable dominant mutants. The ratio of recessive to dominant mutants was 201:l. The average mutation frequency for four known loci was three per 3,172 genomes analyzed. The estimated total number of loci mutated was 535 and the estimated number of kernel mutant loci mutated was 285. Among the 855 kernel mutants, 432 had a nonviable embryo, and 59 germinated but had a lethal seedling. A sample of 194 of the latter two types was tested for heritability, lethality, chromosome arm location and endosperm-embryo interaction between mutant and nonmutant tissues in special hyper-hypoploid combinations produced by manipulation of B-A translocations. The selected 194 mutants were characterized and catalogued according to endosperm phenotype and investigated to determine their effects on the morphology and development of the associated embryo. The possibility of rescuing some of the lethal mutants by covering the mutant embryo with a normal endosperm was investigated. Ninety of these 191. mutants were located on 17 of the 18 chromosome arms tested. Nineteen of the located mutants were examined to determine the effect of having a normal embryo in the same kernel with a mutant endosperm, and vice versa, as compared to the expression observed in kernels with both embryo and endosperm in a mutant condition. In the first situation, for three of the 19 mutants, the mutant endosperm was less extreme (the embryo helped) ; for seven cases, the mutant endosperm was more extreme (the embryo hindered) ; and for nine cases, there was no change. In the reverse situation, for four cases the normal endosperm helped the mutant embryo; for 14 cases there was no change and one case was inconclusive.

C O R N (Zea mays L.) is especially well suited for genetic studies because it has a large pistilate inflorescence (ear) bearing several hundred kernels of suf-

ficient size and complexity to express a wide array of easily observable pheno- Supported by National Science Foundation Grants PCM76105G2, PCM78-08559 to M. G. NEUFFER, and PCM76-

10563 and PCM78485G0 to William F. SHERIDAN. Contribution from the Missouri Agricultural Experiment Station. Journal Series No. 8382.

Genetics 95: 92!3-9-944. August, 1980.

930 M. G. NEUFFER AND W. G . SHERIDAN

types. Many kernel mutants are known in corn (NEUFFER, JONES and ZUBER 1968); one class, largely overlooked for many years because of difficulty in handling, consists of the defective kernel mutants. We have taken a special interest in them because (1) they are one of the larger groups of mutants produced by chemical mutagenesis; (2) they have been practically untouched since the early work of MANGELSDORF (1926) and a few others since; ( 3 ) they affect the development of the embryo as well as synthesis and storage of starches and proteins in the ednosperm; and (4) since they have not been screened carefully, they may be the hiding place of the missing auxotrophs in higher plants (GAVAZZI, NAVA- RACHI and TONELLI 1975; RACCHI et aZ.1978; NEUFFER 1978).

Our intent, as reported here and in subsequent publications, is to examine a large number of defective kernel mutants produced by treatment of pollen with ethyl methanesulfonate (EMS), to investigate and characterize them as a group and to select special classes for intensive study, using biochemical and genetic techniques.

MATERIALS A N D METHODS

The mutants described in this report were induced by treatment of normal corn pollen with EMS in paraffin oil as described by NEUFFER and COE (1977) and screened as described by NEUFFER (1978).

Mutants of all types were identified following EMS treatment, and they were given consecu- tive E numbers beginning with number 1, as well as a tentative description symbol, such as w (albino seedling), cp (collapsed kernel), D (dominant dwarf), etc. Since each MI plant represented 1 complete genome that was subjected to treatment, more than one mutant might be induced in a particular pollen grain. In fact, many selfed M, plants carried several mutant genes, ranging to as many as seven in one case. When an additional mutant was found in the M, progeny of a selfed MI plant, the mutants were designated A, B, (sh-874A, o-874BB, etc.). In the text, only the E number is used to refer to the mutants. Only the kernel mutants will be considered in this report.

Of 855 kernel mutants obtained, 432 contained a nonviable embryo, and 59 germinated but nroduced a lethal seedling. Figure 1 outlines the steps followed in producing and classifying these mutants. Only the most obvious ones were selected for investigation and possible rescue by genetic manipulation as described below and by embryo culture ( SHERIDAN and NEUFFER 1980).

Definitions and nomenclature: In view of the large number of mutants to be described, it was necessary to develop a simple and precise nomenclature. JONES (1920) and MANGELSDORF (1923) called the defective kernel mutants they studied “defective seed” ( d e ) . DEMEREC (1923) designated another class of mutants with nonfunctional embryos as germless ( g m ) . The collection of kernel mutants selected for this study have been termed “defective kernel” mutants in a generalization of the term used by JONES and MANGELSDORF.

Listed in Table 1 are 26 descriptive terms with letter designations used singularly or in combination to describe all the various mutants of the collection. Difficulties arise sometimes because the expression of a particular mutant may change when it is crossed into a different genetic background. Some of these letter designations are the same as gene symbols already in use, but they are used only when the mutant clearly resembles the known gene. As these mutants are identified and located, they will be given an appropriate gene symbol to replace the temporary letter designation.

Heritability of each mutant was confirmed by crossing a selfed plant carrying the mutant onto a standard stock, planting the outcrossed seed, growing and selfing the plants produced to test for genetic transmission. Alexander High Oil (Aho) (ALEXANDER and CREECH 1977) was used as the standard stock in order to transfer the mutants into a vigorous background with a large embryo characteristic.

DEFECTIVE KERNEL MUTANTS I

Treated pollen and crossed on 72 ears 3919 kernels produced

3461 seedlings grew from above kernels 3172 ears produced from selfing resulting plants 855 of these ears segregated for recessive kernel

mutants

* 500 potentially viable

5% or less germinated more than 5% germinated

182 55 39

931

432 21 7 59 147 lethal inadequately tested germinated b u t viable embryo mostly lethal lethal seedling

Sumnary: 855 recessive kernel mutants 432 lethal embryo (355 + 77)

** 217 inadequate test (182 + 35)

147 probably viable 59 germinated, lethal seedling (20 .f 39)

FIGURE 1.-Flow chart indicating steps in production and classification of EMS-induced kernel mutants.

* Separation in this case was made by direct observation. Those ears segregating for drastically aRected kernels with decomposed or missing embryos were classified as nonviable. Those with some semblance of an embryo wem classified as potentially viable.

** These had only one of 20 kernels that germinated to produce a seedling. Many were last because of cultural conditions. Those that survived for progeny test were not hommygous mutant and therefore mus have been either misclassified kemels, pmducts of heterofertilization or from other d o w n causes. Since the other 19 kernels failed to germinate, these were probably lethal cases but tests were not suitable. i Poor test, seedling may have died fmm inadequate growing condhions.

Lethality was determined by planting 20 normal and 20 mutant kernels in a sand bench and checking for germination and seedling growth. A more precise lethality test was conducted by surface sterilizing 20 mutant and 20 normal kernels with 0.53% solution of sodium hypochlorite, placing them on a wet blotter in a petri dish and incubating at 30" for 72 hr or until they had germinated. Seedlings were transplanted to flats and noted for seedling phenotype. Those that grew and died at the seedling stage were recorded as lethal seedlings. We considered a mutant to be lethal if 19 (95%) failed to germinate under test conditions. Lethality tests were done with kernels from the original background and with F, kernels from the progeny of the Grst cross to Aho.

Location to chromosome arm was accomplished by using the B-A translocation method developed by ROMAN and UUTRUP (1952) and described in detail by BECKETT (1978). Each mutant was crossed with a series of B-A translocations in which each arm of the A chromsome set carry-

932 M. G . N E U F F E R A N D W. G . S H E R I D A N

TABLE 1

Nomenclature for kernel mutants

1. bn 2. c l 3. c p

4. crp 5. der 6. de

7. dsc

8. dnt

9. et

IO. p

11. gls 12. gm

13. lsp

14. mn

15. msc

16. nzt

17. o 18. ptd 19. rgh

20. scr

21. sh

22. sml 23. su 24. up 25. u r 26. u x

brown aleurone-brown color in the aleurone cells. colorless aleurone-absence of anthocyanin pigments from aleurone cells. collapsed-the falling in of the exterior endosperm, aleurone and pericarp on an empty interior; giving a thin collapsed kernel. crumpled-surface of kernel like crumpled paper, often opaque. defective crown-crumpled, discolored and/or distorted crown, substantial base. defective-a general term describing an improperly developed endosperm that appears not to be wholly functional and cannot be described by other terms listed here; usually smaller, distorted and otherwise atypical. discolored-some variation of normal clear yellow or white color, as though stained reddish or brownish. dent-small dent in crown area: like dent corn, but segregating on an otherwise nondent ear. etched-surface of kernel with irregular network of pits and fractures of underlying endosperm. floury-endosperm has a soft, chalk-like texture, a generally reduced yellow color and an opaque appearance. glassy-endosperm translucent with a hard flinty appearance, like su2.

germless-embryo very weak o r entirely absent; often associated with a chalky endosperm, a broad flat crown and a pink flush on nonanthocyanin genotypes. loose pericarp-a space separates the pericarp from the aleurone carp, leaving the kernel a greyish appearance over part of the kernel. miniature-kernel typical in shape, form and texture, but smaller in size and with slightly loose pericarp. mosaic-irregular-shaped patches of color and non-color in the aleurone tissue when appropriate genes for anthocyanin are present; the borders are distinct, as opposed to the mottled effect. mottled-irregular distribution of color among aleurone cells with appropriate anthocyanin genes; typically, R mottling. opaque-endosperm is opaque and firm, not chalky and not waxy. pitted-surface of kernel strewn with small pits o r indentations. rough-surface of kernel covered with many small irregularities, giving it a rough texture. scarred-surface of kernel with patches of defective tissue, often joined in an irregular pattern. shrunken-two types: (1) a moderately smooth collapse of the crown and sides of the kernel, like shl, and (2) an extreme collapse of endosperm tissue, often associated with a brittle and/or translucent appearance, like sh2. small-used in conjunction with other descriptive terms to designate smaller size. sugary-kernel is shrivelled, hard and translucent; typical of S U I . viviparous-premature germination, coleoptile elongated. wrinkled-kernel appears full, but surface has long, irregular identations. waxy-endosperm hard an6 opaque with a waxy texture; the cut surface stains red with IKI.

DEFECTIVE KERNEL MUTANTS I 933

ing nonmutant genes is translocated to a centromere carrying segment of a B chromosome. Thus, we have a reciprocal translocation stock for the short arm of chromosome I with the shofl arm of 1 attached to the centromere carrying region of a B chromosome and the long arm of i with its centromere attached to an acentric B chromosome region. The same is true for each of the other arms in the A chromosome set. Characteristic nondisjunction of the B centromere at at the second microspore division produces sperm nuclei with either 2 sets (duplicate) or none (deficient) of the particular A arm attached to the B centric region. If each of the whole set of B-A translocations is crossed as pollen parent on ears carrying a recessive mutant, the ear crossed by the B-A chromosome involving the arm on which the mutant is located will produce some progeny that will lack the male contribution for that segment and will therefore be hemi- zygous (m-) for the mutant. Since the mutant gene will be uncovered, it will be expressed and its arm location thereby identified; in all other crosses the progeny will be heterozygous (m+) and nonmutant in appearance (Figure 2). Tests in this study were made by planting 75 normal kernels from a selfed ear that segregated for the mutant to be tested. Then, since two-thirds of the plants grown should be heterozygous for the mutant, 3 ears were crossed by pollen of each of the 18 B-A chromosome translocation stocks representing the 18 of 20 maize chromosome arms that are available in useful B-A translocation combinations. The B-A chromosome set was supplied by J. B. BECKETT.

The resulting ears were examined for c a w showing substantial numbers of mutant kernels. As stated above, these would result from a cross of a heterozygous ear parent (+/m) by a pollen parent carrying the B-A translocation involving the same chromosome (+/B-A+). The mutant kernels would then appear when the polar nuclei containing the mutant gene were fertilized by a

Parents

Gametes

F, kernels

00

defec t ive embryo

x

defec t ive endosperm

non-disjunct ion

normal kernel

FIGURE 2.-A schematic representation of genotypes and kernel characteristics of the parents and F, from a cross of a plant heterozygous for a kernel mutant (+m) , by a plant carrying the B-A chromosome translocation with the normal allele at that locus in the A chromosome arm segment. Only progeny receiving the m allele from the female plant are shown. This is termed the arm-locating cross.

934 M. G. NEUPFER AND W. G. SHERIDAN

Endosperm (top) Embryo (bottom) constitution

+ + + m m m m m - m m + m m + + + + m m m + + m + m - 1 2 3 4 5

FIGURE 3.-Photograph of longitudinally split mature normal and mutant kernels (I and 2) from a selfed ear of a plant heterozygous (+m) for clf (E79.2) and mutant endosperm (mm-) nonmutant embryo (m++), kernel 3; nonmutant endosperm (mm+) nonmutant embryo (mi - ) , kernel 4; nonmutant endosperm (mm++) mutant embryo (m-), kernel 5; kemels 3,4 and 5 are from an ear produced by the class of a heterozygous em parent by pollen carrying the translocation that uncovers the short arm of chromosome 1.

speml nucleus that was deficient for the t h m ” e arm on which the mutant was located. The other 17 B-A translocation crosses would produce only normal kernels.

Endarperm-embryo interaction studies were carried out with material produced by the ann- locating cross of the B-A translocation series described above. Some of the phenotypically mutant kemels on these ears have hypoploid mutant ( m m ) endosperms associated with hyperploid normal (m++) embryos. In addition, there should be an approximately equal number of kernels with hyperploid normal (mm++) endosperms associated with hypoploid mutant (m-) embryos. Finally, the majority of kernels should be normal because they received a normal allele either from the heterozygous female parent or from the B-A translocation male parent.

Typical examples of each kernel class were cut longitudinally to observe distinguishing characteristics of their respective endosperms and embryos. For comparison, normal and mutant kernels from the selfed progeny were handled in a similar fashion (see Figure 3). In addition. a 50-kernel block from the B-A cross was separated into the three kernel classes, which were then planted in a sand bench to test for viability and growth characteristics in comparison with a similarly separated sample from a segregating selfed ear.

RESULTS

Seventy-two ears were pollinated with EMS-treated pollen; 63 set an average of 62 kernels, with a range from 5 to 230 (Table 2). Twenty control ears had an average of 248 kernels. The 3,919 kernels produced from treatment were planted in the field, along with 637 control kernels.

In the treated material, 3,461 of the kernels produced seedlings. Of these, 16 had a mutant phenotype (distinguishable from normal and from monosomics, haploids, etc.). These were classified as dominant mutants. Twelve of the 16 were lethal. The remaining four proved to be heritable dominant mutants. Most of the seedlings grew to mature plants that were selfed to produce 3,172

ears. A considerable number (85 1 ) of these ears had scattered seed sets of the type characteristic of semisterility that occurs as the result of failure of transmission of


TABLE 2

Total heritable changes induced by treatment of pollen with ethyl methanesulfonate

Dominant Dominant Semi- MI % lethal viable Selfed sterile

Treatment seed plants plants plants ears ears

EMS 3919 3461 12 4 3172 851 % 88 0.3 0.1 81 27 Control 637 575” 0 0 451* 1

Heritable changes 2477/3461* Recessive mutants 2457/3172** Recessives less semi-sterile 1606/3 172 Recessive kernel mutants 855/3172 Viable dominants 8/3461 Ratio recessive/viable dominant 1606/8 Average frequency of four known loci 3/31 72 Estimated loci mutated 1606/3 Estimated kernel mutant loci 855/3

% 90 <0.16 <0.16 - 0.16

Dominant Reces$ve Recessive Total endpsperm seedling kemel heritable mutants mutants mutants changes

4 751 855 2477 0. I 24 27 63 0 3 0 4

<0.16 0.52 0 0.7 0.716 0.775 0.506 0.270 0.0023 eoi/1 0,00095

535 285

Frequencies are expressed as percent of M, seed tested, MI plants grown or selfed ears tested, as they apply in each case.

* 3461 is the actual number of individuals that were examined for heritable changes-note that this includes the screening for dominant mutants; however, not all 3461 plants were selfed.

** 2467 1s the number obtained when the dominant mutants (20) are subtracted from the total heritable changes (2477).

a chromosome aberration. (No attempt was made to distinguish these from ones due to poor pollination.) The 3,172 ears on self4 MI plants included 859 that segregated for distinguishable kernel types. Four had approximately 3 mutant: 1 normal kernels and were classified as dominants. The remaining 855 ears segregated approximately 3 normal kernels to 1 mutant type kernel and were classified as recessives.

Decetiue kernel mutants: The recessive kernel mutants found were grouped into four types: Type 1 those affecting the endosperm and embryo but with a nonviable embryo; type 2, those affecting both the endosperm and the embryo and having a viable embryo that produces a seedling with a distinctly mutant phenotype; Type 3, those affecting the endosperm only and therefore capable of producing a normal seedling; and Type 4, those affecting the embryo only and expressed as “germless” kernels.

In many cases, the embryo of Type 1 mutants was already necrotic by the time the kernel had matured. These types were of interest because they affected the development of the embryo at some stage between zygote formation and kernel maturity (SHERIDAN and NEUFFER 1980).

Seedling phenotypes of Type 2 mutants included white, yellow, yellow-green, distorted, dwarf, glossy, necrotic and miscellaneous small and weak types. Many mutants characteristically expired soon after emergence, some continued to grow as weak seedlings for several weeks before dying, some died as soon as the limited endosperm reserves were depleted (about two weeks after germination and a few grew to maturity.

936 M. G. NEUFFER AND W. G. SHERIDAN

Type 3 mutants were similar and often allelic to known mutants such as shrunken, sugary, opaque, waxy, etc. Generally, but not always, the plant phenotype was not appreciably different from normal.

Type 4 mutants included those that were described as germless by DEMEREC (1923). These mutants appear in the kernel class because, in contradiction to our definition above, the absence of a normal embryo often causes a recognizable dis- tortion of kernel shape and/or a discoloration of the endosperm of some mutant kernels on a segregating ear. Most were not found among the original 855 mutants in Table 2, but were recognized later in the more detailed examination of proge- nies segregating for mutants of Types 1 to 3. A careful reexamination of all the Mz ears would no doubt reveal more of the “germless” type.

The original 855 kernel mutants (Figure 1) included 432 Type 1,59 Type 2, 147 Type 3 (viable embryo-normal seedling) and three Type 4. The remaining 214 could not be properly classified because they did not receive an adequate test. Most will probably be lethal and will be added to Type 1.

Selection was made among Type 1 mutants for the full range of most readily recognizable phenotypes to study in detail. A total of 196 defective kernel mutants were examined (Table 3) ; these included 194 EMS-induced cases and two spontaneous proline-1 mutants (P~O-GAVAZZI and pro-342, COE) . The latter two were included for comparison. Of the 196 mutants, 150 had easily distinguishable defective endosperms, displayed segregation patterns on immature ears, and had nonviable embryos or lethal seedling phenotypes in their original genetic background. Varying amounts of information is available about the remainder, and they are included in Table 3 for that reason. By limiting our tests to the clearest cases, we eliminated many problems of classification.

Chromosome mapping data: Of 396 kernel mutants examined in these experi- ments, 165 were located to a chromosome a m on the basis of a first test. A 42% location rate may appear low when one considers that the 18 translocations cover an estimated 85% of the genome (BECKETT 1978). A number of factors reduced the coverage of each test, including the fact that most of the mutants are lethal, which necessitated the use of heterozygous plants, as well as limitations in availability of pollen from translocation stocks.

Of the 194 EMS-induced mutants listed in Table 3, most have been tested for location to chromosome arm and 89 have given one positive location. Of these, 39 have been confirmed by a second cross. The distribution of these locations is presented in Table 4, with the italicized entries representing the confirmed locations. The distribution frequency correlates well with a m length when adjusted for some complicating factors, namely: (1) because of vigor and availability of pollen, some arms (i.e., I S , 2s and 5 L ) received a more complete test than others (5S, 68 and 9 L ) ; (2) some arms are not well-covered because of the limited extent of the translocations (5S,6S and 8 L ) j and (3) in some cases the expression of the hypoploid endosperm mimics the defective phenotype to some degree (5S, 7L and IOL) . The use of these latter three translocations results in hypoploid endosperms that are small and/or opaque and defective. The locations for these three arms were initially inflated by false cases, but then much reduced by eliminating

DEFECTIVE K m N E L MUTANTS I 937

TABLE 3

List. of 196 defective kernel mutants used in this study

E. No.

* 76B * 198C

211c 330D 627D 628A 660C

* 660E 740 744 74 7B 749 788 792

* 863A 8 73 874A 874B 883A 888A

890A 901A 912 918A 923

* 924 925A 928A

* 928B 931A 932 933

* 935 936A 948A

971 974A 991

1005A 1007A 1009 1022

* 889

* 948B

Kernel p h e n o t w

C P dnt w r sh su

C P

o sh C P

de ptd fi!

de o bn o

dsc et

Cl P C P

C P

sh 0

0

crp dsc dnt o

mn dsc ptd

sh C P 0

Ptd

dcr 0

cl P iTm

C P 0

de 0

C P

C P

C P

sml o CP 0

wh C P

dcr de

C P 0

de

Viability or seedling phenotype1

0 0

gs 0 0 0 + + 0 0 0 0 0 0 0

w-gs

-

W + + + a

+ + +

-

gs

0

0 0 0 0 0 nl 0 0 d + + 0 +

nl + tY

Arm location E. No.

Viability Kernel or seedling Arm

phenotype+ phenotype$ location

4s 3L 3L

2s 3L

9L

7L I S

9s 3s

4s

6L 5s 3L is

4L

*lo32 1045 1053 1054 1058 1060A 1060B

*1069A 1076A

'1078A 1078B

* 1079 1089 1092A 1092B 1096A 1104B

*I105 1112 11 13A 2119A 1121 1122A

*1126A 1130

*1132 1142A 1145A 1147A

1154A 1155A 1156A

*1156B 1157

*1160 1162 1163

*1166 I1 67A

*1167B 1168A

*1172A 11 72B

*I153

o rgh lSP

dcr dsc

C P

crP C P

o rgh de C P

et C P

o sml de C P

et o sml

de rgh rgh C P

o sml CrP de de Pt.d

de o de

P dnt. sh de

P r s h de o

sh dcr sh sml CP

P de sh sml sml 0

C P

- 1 + 0

0-gs 0 - - 0 + + 0 + 0

+ -

- W + 0

0 -

+ + 0 + 0 0 0 + 0 0 - - + + 0 0

0

0

-

-

+ -

3L

8 2s

4L

I S

4L

5L

IL

938 M. G. N E U F F E R A N D W. G. SHERIDAN

TABLE 3-Continued

Viability Viability Kernel orseedling Arm Kernel or seedling Arm

E. No. phenotypet phenotmS location E. No. phenotype+ phenotype$ location

1024A 11 76A

*1176B 11 77A 1180A

*1189A 1191 1196 1202A 1208A 1210 1225A

*1225B *1230 * 1233A 1239 1241 1253B 1255B

*1275A 1283 1286A 1287

*I289 1294 1295 1296A

*I298 1299 1303 1308A 1309A

*1310A 1310B

*1311A *1311B 1311C

*I312 1313 1315A 1316A 1319A

*1319B 1324A

'1328A 1330

CP P 0 CP 0 der - d e - de +

o sml a d e 0 d e 0 P 0 P + r g h 0 P 0 CP snil - dcr 0 CP 0 0 1 fl 0 CP 0 CP 0 CP 0

CP 0 ,+

P 0 gm 0

c p dsc 0 sh 0

CP 0 0 dnt o 0 f l dnt 0 P gm 0

CP 0 P d n t 0 o sml +

d e nl o sml 0

gm 0 CP 0

gm 0 0 0 0

dsc scr 0 P + CP d P n 0

sh ptd 0 sh +

-

m S C 0

2L 6L

IS

7L

3s

2s

6L

5L IL IS 9L

3L

IL

5L IOL

1175 d e 13 73A sml 1379A CP 1380A CP

*1380B o sml 1381 CP 1383 0

1384A o sml 1385 CP 1386A d e 1387A CP 1388 o sml 1390A de

* 1390B .fl * 1390C gm 1391 P 139249 CP 1393A CP 1394 cl fz 1395A d e

*1396A d e sml 1399A CP 1401 cl P 1404 CP 1405A CP 1406 CP 1409 d e 1410 CP 1411 sh 1413 d e 1414 f l 1415A CP 141 7 d e 1418 CP 1419 C P

14.20 d e 1421 s h 1422 0

1423 CP * 1424 o yml 1425A dsc ptd 1426 P 1427A CP P 14828 dcr 1429A CrP 1430 CP

- 0 0 0 0 0 - + 0 0 + 0 0

0 0 0 0 0 I S 0

0 0 15 0 0 0 0 91 0 0 + 0 21 0 0 IOL 0 0 0 0 0 0 0 0 0 0 IOL 0 0 0

-

-


TABLE 3-Continued

Viability Viability Kernel orseedling Ann Kernel or seedling A m

E. No. phenotypet phenotype$ location E. No. phenotypet phenotype2 location

1331 1332 1333B 1339A 1341 1348 1365 1369

C P

et P

et sh et sh cl P

CrP CP

0 0 0 W + 0 0

nl

5L 1431 7L 14.35

3s *I479 * 1484

IS '1485 5L pro-I 5L pro-342

1436A

194 are EMS-induced and two are spontaneous proline-I mutants (pro-GAvAzzI and pro-349, COE). Each is listed by E number with kernel phenotype, viability or seedling phenotype (if kernel germinates) and chromosome arm location where confirmed.

* Not included among 150 used for genetics and embryo cultures rescue attempts (SHERIDAN and NEUFPER 1980) because mutant produced a viable normal seedling or because its phenotype was not easily recognized early enough for embryo culture. t 0, nonviable, failed to germinate; +, normal green seedling; -, no test.

d, dwarf; gs, light green stri es; 1, luteus yellow seedling; nl, narrow leaf; pg, pale green seedling; ty, tiny; w, white seedfing ; w-gs, white with green stripes.

f See Table 1 for explanation of symbols.

all those that could not be verified. Since confirmation for the mutants located on arms that produce false cases is so complicated and the numbers involved so large, possible false cases were excluded; only those few that were readily distinguishable from the hypoploid conditions are included in Column 8 of Table 3.

Endosperm-embryo interaction: The role of the developing endosperm in the development of the immature embryo can be considered by comparing the response of the three classes of kernels (obtained on the ear that revealed the chromosome arm location when a successful test was conducted) with the response of concordant mutant kernels (with mutant embryos and mutant endosperm) from a self-pollinated heterozygous plant. Reference to Figure 2 shows that these three classes of kernels are: (1) kernels with hypoploid mutant embryos and hyperploid nonmutant endosperms; (2) kernels with hyperploid nonmutant embryos and hypoploid mutant endosperms; (3) kernels with normal embryo and endosperms.

Preliminary results are shown in Table 5 for 19 mutants. In three cases (EZIIC, E330D and E627D), the mutant endosperm was less extreme in expression than in the concordant mutant. In seven cases (E792, E928A, E1289, E1299, E1303, E1311B and E1348), the expression was more extreme; in the remaining 19 cases, the expression was like that of the concordant mutant.

An example of the second or more extreme type of expression is shown by comparing longitudinally split kernels from selfed and B-A crossed heterozygous ears for the mutant E792 (Figure 3). The mutant located on the short arm of chromosome 1 and designated colorless floury-I (cy) causes failure in the formation of anthocyanin pigment in the aleurone and carotenoid pigments in the endosperm and also failure of embryo development. Note the white, fluory, germless, concordant mutant kernel (second from left of Figure 3) . The mutant

940 M. G. NEUFFER AND W. G . SHERIDAN

f d - %E%:% " W * Y

h - h

g E $ - 0- - h

Y - q Q \ o ,

k W W * h h -

-a.-

f C d Y - m - 0 1

g % g $ - - *

$ 2 h E

B 8 % ' - i c y ; 2 h . o W W

2 E 9 W M W * \ c e $ $ j z g Q g 2

% h * h

u 20(0hoohtnn0\0 % : ! ( D \ o O , h c y W \ o W o \

p\ Q " \ '-i'-i

f f

0 f L9

8 m ' - i , " h l * K \ n \ m r * ,

\.-h'-i'-ih'-ib U

* $ O w : ' D q Y

CA

w s f f 4 g ! $ m o m

$ 3 '-i-h

.$ ?&g C

g m t n --i

Q o \ O ,

Q Q C m Q 2 3 g z $ g z g : 2 "\\c'ao-m, 4 -3 , ; : c o , . r , 6 \ % m - g - w -

.U

h + h

m f f f i -t

e-3- - h h

M c1

- w U u o $ 2 m n % v . a , N .2 i g y & ; $ $

h d - - ' - i * f ffi4

w L4 ffi c $ 2 3 o\ c.1 M M (0

M W h m a h m Q M r o M * ~ $ z ? h Q M c o * I m m

2 & m w d w c y c o c o % % ' - i T C t T f 8 2 3 2 8 8 : 8 % ;r: Ma 2 9

$ g 3

h(C(h*-.-+d

C C 4 C f T t! % 2 % 2 2 2 2

~ 0 a m - h ~ ~ W h h h

- h h ' - i h - h b

a,

E

d= \ c B id

Eau, 2% E!& ti$!

: .5 ; E Z6 + 'F $ 2

5E 0 5 2-2s 2.6 - - 0 g w

'U .s

P * + d , w d

+d

.-a c *

a - .

m a ) h >a .+ p:

H :$ 9 @# s h-0

- f i c-3 m 8 2 0

P m 0 auw" $2 n

.G 2G z % --

.* $ 2 3 c-= 4 '- $0 D z- 0- U 4 m l s l ? fi: z m b f 2 2 h P, .a ul+ 8 g o >

h g

2 - N

P

+ + - a .d

. 3

g z g 75 g.5 '3 6 e: a $5 5 2.5 3g;;; & z 2 %ae .so1 a zz E m . r % & .c s o $34

wit

om M

a+a)

c c

U,%

!z


TABLE 5

Listing of 19 defective mutants tested for endosperm-embryo interaction in noncorresponding hyperploid-hypoploid combinations from the cross of +/mutant by B-A translocation

of the proper constitution

%?Y&% Phenotype mm++ endo

Vibilitv. m- embrvo m++ embmo Mutant Chromosome concordaiit- - --- E. No. ann . mutant Embryo Seedling Endosperm Embryo

(1) (2) (3) (4) (5) (6) (7)

211c 330D 627D 792 873 928A 1122A 1160 1208A 1283 1289 1296A 1299 1303 1311B 1315A 1331 1332 1348

4L 3L 3L 1s 9 s I S 2s 5L I S 3s 2s 6L 5L IL 3L IL 5L 7L I S

gs 0 0 0 f? 0

weak + nl 0 0 0 0 0 0 0 0 0 0

N m N m N? m m m N m m m m m m N m m m

gs 0

leth 0

leth 0

leth 0 N 0 0 0 0

leth 0

Y g 0 0 0

t

t

t -+

4

c

-3

3

--f

4

N N N N

weak N

tiny N N N N N N

weak N

leth N N N

Mutants are listed b E number (column 1) and arm location (column 2). Viability of the concordant mutant (COL- 3) is expressed as 0 for failure to germinate, a phenotypic symbol (e.g., gs) for those that germinate but roduce a lethal seedling, a + for those that germinate and produce a normal seedling. The pfenome of the mutant embryo-nonmutant endosperm class of kernels (Table 4) i s expressed either as m (visibly mutant embryo) or N (normal embryo). In the latter case, this class could not be distinguished, so that all normal kernels were planted and identified by mutant seedling phenotype if they grew or by lethality if they failed to grow. These results are combined with those from planting the distinguishable mutant embryo types and are expressed (column 5 ) as 0 for those that failed to germinate, leth. for those that germinated but died immediately, and a henotypic symbol (gs or yg) indicating those that produced a seedling with a distinguishable pienotype.

The phenotype of the nonmutant embryo but mutant endosperm class of kernels (columns 6, 7) is expressed (column 6 as a mutant endosperm that is either more extreme (-), the same as (m) , or less extreme (4) than that of concordant mutant kernel and as nonmutant embryo (column 7) that appears as normal (N) and grows into a normal seedling, as a weak seedling, as a definitely inhibited seedling or as a lethal. The arrows between columns 6 and 7 indicate the direction of advantage in the feeding relationship. For example, for mutant E792, the arrow pointing toward the N indicates that the endosperm is more severe in its phenotype (presum- ably because of the nutrient demands of the developing normal embryo) than was observed in the concordant kernel, wherein both tissues are mutant.

endosperm-nonmutant embryo kernel (third from left, Figure 3) has mutant endosperm that is white, floury and much reduced, indicating failure in pigment and normal starch formation, but it has a large, almost normal embryo. Two things are clear: the normal embryo did not contribute to anthocyanin, carotenoid or starch development and the developing normal embryo either competes with or actively depletes the developing mutant endosperm. The mutant embryo-

942 M. G. N E U F F E R A N D W. G. SHERIDAN

nonmutant endosperm kernel (far right, Figure 3 ) has an endosperm with fully developed anthocyanin, carotenoids, corneous starch and nearly normal volume, but contains a tiny embryo. Clearly, the endosperm provided no help for embryo growth in this case.

When kernels with mutant endosperm and nonmutant embryos from these 19 cases were planted, they produced normal green (though some were somewhat less vigorous) seedlings in all but two cases. El 122a produced a tiny weak green seedling that grew slowly and lacked vigor even at six weeks when the experiment was terminated. E1315A produced an abnormal seedling that wilted and died soon after emergence.

From these results, we concluded that during kernel development: in three of the 19 cases, the normal embryo helped the mutant endosperm towards normality, in seven cases the normal embryo depleted the mutant endosperm and in nine cases there was no appreciable interaction; conversely in 17 of 19 cases there was no affect of the mutant endosperm on the normal embryo; in one case the mutant endosperm considerably weakened the normal embryo and in one case the mutant endosperm caused the death of the normal embryo.

It was readily apparent by visual examination of 14 of the 19 mutants tested that those kernels containing a mutant embryo and a nonmutant endosperm were identifiable. These are designated “m” in column 4 of Table 5. All except El 122A appeared as typically “germless” kernels since they had only tiny remnants of a dead embryo and failed to germinate. El 122A did have a tiny weak embryo that germinated but died without further development. In all 14 cases, we concluded that the genetically normal endosperm did not help the mutant embryo.

For the remaining five mutants, the ears produced by the arm-locating cross had no distinguishable mutant embryo-nonmutant endosperm class (visual examination revealed no kernels with a normal appearing endosperm and obvi- ously reduced embryo). From these ears, normal kernels were randomly selected for planting to test for mutant seedlings. If the mutant embryo were aided during its development, but not permanently, then its mutant condition should become apparent upon germination. However, if the mutant condition affected only em- bryonic development and not processes during or subsequent to germination, then normal appearing seedlings should be produced by all tested kernels. In two cases (E627D and E873), all kernels germinated, but a portion of the seedlings died soon after emergence. These were assumed to contain mutant embryos whose phenotype was seedling lethality. In two cases (E21 1C and E1315A), all kernels germinated, but a portion produced distinguishable mutant seedlings (gs and yg, respectively). These had grown almost to maturity when the test was ended. Fi- nally, one case (E1208A) produced all normal seedlings, so that the mutant embryo class was not recognizable; however, this was not unexpected as this mutant was the only one that had a viable embryo in the concordant mutant.

We conclude that four of these remaining five cases, with ears that had no distinguishable mutant embryo class, were helped to some degree by normal endosperm, at least to the extent of producing a visibly normal embryo, Two (E627D and E873) failed after some growth, and one (E1315A) produced viable, though


clearly mutant, plants (E1315A) and one (1208A) produced normal appearing plants. The remaining case (E211C) was not a lethal mutant in the first place, but produced a green-striped seedling.

DISCUSSION

Number and variety of mutants: We were successful in producing a large collection of new mutants using the paraffin oil technique for treating pollen with EMS. The data presented in Table 1 show that from a population of 3,461 MI plants, a total of 2,477 heritable changes were produced (72%). A large propor- tion of the mutants (855) were found to be kernel mutants; however, we antici- pate that future screening of this collection will identify many additional mutants, including “germless kernel” mutants and mutants affecting mature plant characteristics.

The question is: How many loci are represented in this mutant collection; are there many repeats of a few loci or many new loci? As pointed out by NEUFFER (1978) from calculations made from the identification of four selected, easily recognized known loci, the average frequency of recessive mutation per locus in this collection was 3/31,972, or 0.9 per thousand gametes. The 1,606 recessive mutants found in this experiment were calculated to represent 535 loci. Following this same reasoning, we may conclude that the 855 kernel mutants found represent approximately 285 loci.

Distribution of Zoci: The data in Table 3 show that the mutants are distributed on 17 of th 18 arms tested by theB-A translocation method. Since the translocation stock for chromosome arm 6s uncovers only a small portion of that arm, failure to locate mutants to that arm is not significant. On the basis of the above results it is apparent that the mutant loci are scattered throughout the genome. Furthermore, assuming the presence of 285 nonallelic mutants, there are on the average about 14 mutant loci per chromosome arm. At the present time, we are not able to estimate how many loci are represented among the 89 mutants listed in Table 4 because we have not yet undertaken any extensive testing for allelism.

One group of Type 1 mutants, appearing as thin, paper-like empty shells resulting from the early failure of both embryo and endosperm development, did not give positive results in arm location tests. Their developmental fate is cur- rently being investigated by histological methods.

Endosperm embryo interaction: The results of the study on endosperm-embryo interaction indicate that in the majority of mutants, a normal endosperm is un- able to rescue the development of a mutant embryo, although in some cases rescue does occur. Even in such instances, however, the mutant embryo is not permanently rescued, but dies (if carrying a mutation that is lethal in concordant kernels) following germination and depletion of endosperm reserves. These obser- vations, although supportive of the nurturing role of the endosperm during germination, strongly indicate the dependency of the developing embryo on its own genome (and tissues) for its growth and development.

944 M. G . NEUFFER AND W. G . SHERIDAN

Technical assistance from KAREN SHERIDAN, JOE SHEA and MARGARET HOBAN is gratefully acknowledged. We also appreciate the help of MING TANG CHANG for illustrations, CINDY ROY and PATRICIA ESTEPP for the stenographic work, and especially EVELYN BENBOW for able assistance in all aspects of the study.

LITERATURE CITED

ALEXANDER, D. E. and R. G. CREECR, 1977 Breeding special industrial and nutritional types. pp. 363-390. In: Corn and Corn Improvement. Edited by C. F. SPRAGUE. American Society of Agronomy, Inc., Madison, Wisc.

BECKETT, J. B., 1978 B-A translocations in maize. I. Using in locating genes by chromosome arms. J. Heredity 69 : 27-36.

DEMEREC, M., 1923 Heritable characters of maize. XV. Germless seeds. J. Heredity 14: 297-300. GAVAZZI, G., M. NAVA-RACHI and C. TONELLI, 1975 A mutation causing proline requirement in

maize. J. Theor. Appl. Genet. 46: 339-346. GAVAZZI, G., G. TODESCO and M. G. NEUPFER, 1978 Location of the pro mutant to chromosome

arm. Maize Genet. Coop. Newsletter 52: 66-67. JONES, D. G., 1920 Heritable characters of maize. IV. A lethal factor-defective seeds. J.

Heredity 11: 161-167. MANGELSDORF, P. C., 1923 The inheritance of defective seeds in maize. J. Heredity 14: 119-

125. -, 1926 The genetics and morphology of some endosperm characters in maize. Conn. Agr. Exp. Sta. Bull. 279: 509-614.

NEUFFER, M. G., 1978 Induction of genetic variability. pp 000-000. In: Genetics and Breeding of Maize. Edited by D. B. WALDEN. Wiley-Interscience, New York.

NEUFFER, M. G. and E. H. COE, JR., 1977 Paraffin oil technique for treating mature corn pollen with chemical mutagens. Maydica 23: 21-28.

NEUFFER, M. G., L. JONES and M. S. ZUBER, 1968 The Mutants of Maim. Crop Sci. Soc. Amer., Madison, Wisc.

RACCHI, J. L., G. GAVAZZI, D. MONTI and P. MANNITO, 1978 An analysis of the nutritional requirements of the pro mutant in Zea mays. Plant Science Letters 13: 357-364.

ROMAN, H. and A. J. ULLSTRUP, 1952 The use of A-B translocations to locate genes in maize. Agron. Jour. 43 : 450-454.

SHERIDAN, W. F. and M. G. NEUFFER, 1980 Defective kernel mutants of maize. 11. Morphologi- cal and embryo culture studies. Genetics 95 : 9 4 - W .

Corresponding editor: R. L. PHILLIPS

defective of maize. i. - home | genetics · 2003. 7. 29. · defective kernel mutants of maize. i....

Documents