REVIEWS

Chromosome engineering of pollen wheat

HU Han, ZHANG Xiangqi, ZHANG Wenjun, JlNG Jiankang , WANG Erming
and WANG Xianping
St& Key Laboratory of Plant Cell a r t Chromosom Engineering Institute of Genetics,
Chinese Academy of Sciences, Beijing 100101, China
Abstract Chromosome engineering of pollen wheat is the new procedure combining anther cul-
ture and chromosome engineering techniques. It could transfer useful alien genes into wheat
varieties, enhancing genetic diversity for investigation of genetics and breeding. In the present
study, two new procedures, at genome level and single chromosome level, were established.
Compared with the classical chromosome engineering, the chromosome engineering of pollen
wheat has the following main characters: ( i ) diversity of products, ( il ) rapid stability and
( iii ) high efficiency of selection. Experiments indicated that chromosome engineering of pollen
wheat is an efficient way for creating alien translocation line, especially non-Robertsonian
translocation line. Meanwhile, using this procedure combined with comprehensive identifying
methods, the investigation of useful genes and molecular markers on rye chromosomes 1R and
6R respectively has been done. The roles and relationships between chromosome engineering
of pollen wheat and gene engineering on crop improvement were discussed.
Keywords : wheat, anther culture, chromosome endneering, translocation, gene mapping.

CHROMOSOME engineering is the method and technique designed to reduce, add and substitute homological
or alien chromosomes according
.-
to plan. It could be named chromosome manipulation that is the cell engi-
neering at chromosome level1" . Its basic procedure is: firstly crossing, secondly producing gametes with
different chromosome composition (number and structure) based on the usual variation of chromosome
synapses during meiosis, and thirdly screening through cytological identification in hybrids or progenies of
hybrids.
In recent years, due to the poor resource of genetics and germplasm of interspecific wheat, variety
improvement has been seriously influenced. However, alien wheat species genera, such as Secale ,
Aegilops , Elytrigiu , Hynaldia , possess useful genes related to productivity, good quality, resistance to
stress, etc . Therefore, investigating and utilizing alien genes, and transferring them into wheat varieties
to enhance genetic diversity is an important project to improve wheat genetics and breeding. But the tradi-
tional wide hybridization and chromosome manipulation procedure is quite complicated and time-consum-
ing, and it needs skill cytological technique and knowledge.

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1 Principlesand procedures
( i ) Basic principles of chromosome engineering of pollen wheat. Haploid pollen possess only
one set of chromosomes with special genetic characters: ( 1 ) Pollen clonal variation is easily produced
during anther culture in vitro. This kind of variation possesses universality:'-41 . 'Ihere is variation in the
number as well as in the structure of chromosome^'^: , and the main mechanism for chromosome structure
variation is breakage and reunionL6'. These principles paved a new way for producing translocation lines
and gene mapping. ( 2 ) There are no sexual processes in anther culture in vitro. So the competition of
gametic types in fertilization was avoided, and different types of gamete might be fully kept, including re-
combinants and variants, which could be fully expressed in one generation and rapidly stabilized at pollen
plant level. Meanwhile the efficiency of selection could be increased, and the cycle of investigation and
breeding for engineering materials was greatly shortened. Yew wheat gennplasm with target genes or chro-
mosomes could be obtained within about two .
( ii ) Procedures of chromosome engineering of pollen wheat. Chromosome engineering of pollen
wheat is the new procedure combining anther culture and chromosome engineering techniques. Its basic
procedure is using the Fl hybrids between secondary distant hybrids and common wheat as the materials
for anther culture; in one generation alien haploids and homozygous alien diploid with diversity of genetic
composition could be obtained. Then advanced and comprehensive techniques are used to rapidly and
exactly screen the new germplasm of chromosome engineering.
In the experiment, we improved the traditional chromosome engineering method and established two
new procedures as follows: (1 ) Procedure at genome level. This procedure started to be used in the early
1980s. Using secondary distant hybrids hexaploid tdicale ( AABBRR) to cross with common wheat
(AABBDD), we obtained FI hybrids (AABBDR) with two D, R haploid genome; or using octoploid
Elytrigia (AABBDDEE) to cross with common wheat, we received septaploid F, hybrids (AABBDDE) .
These FI hybrids were used as materials for anther culture. According to the principles that the haploid
gametes can be fully expressed and doubled haploitis are stable in one generation, a lot of new
gennplasms with different alien chromosome compositions were obtained in a short time, and the quantita-
tive distribution coincided with the theoretical value. Some types of them were difficult to obtain by con-
ventional crossing methodsrs1; (2) procedure at single chromosome level. Two kinds of procedures were
established. First, we used substitution or addition lines with object chromosome to cross with common
wheat or proper wheat deletion line to cross with common wheat and F1 hybrids for anther culture. HI pro-
duced aneuploids with object chromosome or chromosome fragments (arm) ; for example, ABO 6A nulli-
somic was crossed with M24 (6R/6D) substitution line. The F1 hybrids were used for anther culture and
various alien aneuploids were obtained, including 6R addition, 6~~ telomere addition, deletion addition,
isoarm addition, etc. This alien aneuploid population was good for gene mapping. The other procedure
was using hexaploid triticale 8 pin-17 to cross with Chinese nulli-tetra N6D-%A corresponding with ob-
ject 6R chromosome; F1 anther culture was canied out, and at HI aneuploids with object chromosome
were produced. ZH-1 ( 2 n = 44) is a 1R addition and 6IW6D substitution line obtained by this p m e -
dure. From crossing to identification of genetic composition, the process only took 18 months (June,
1992-November, 1993)'). There are two main advantages of chromosome engineering of pollen wheat at
.
single chromosome level : First, using single chromosome ( e . g 6 R ) for transfening clear object genetic
composition, genetic analysis can be easily carried out. Only object chromosomes and appropriated groups
of wheat need to be analyzed. Second, object chromosomes can be rapidly transferred. By this procedure
different p d u c t s with object chromosomes could be obtained within 1.5 to 2 years. Since the beginning
of the 19909, by using the two new procedures mentioned above, genetic compositions of more than 4 0
pollen plant lines have been identified (table 1) . The facts indicated that the improved chromosome engi-
neering procedures greatly increased the efficiency of research work for application.

1) Zhaq, X .Q . , Summary of postdoctoral research work, 1993.

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Table I Alien anruploid of pollen plant lines in wheat

Ty~e Nuhr
Alien translocation line 10
Alien substitution line 13
Alien addition line 12
Alien multi-addition line 5
Alien addition-substitution line 2
Total 42

( iii ) Characteristics of chromosome engineering of pollen wheat. ( 1 ) Diversity of products.

Since the gamete selection was reduced in anther culture, more recombination and variation types than
sexual progenies could be obtained by this procedure in small populations of pollen plants, not only the
general products of chromosome engineering could be directly obtained, such as addition line, substitu-
tion lines and translocation lineL9],but also more complex new types of genetic composition could be easi-
ly produced which are difficult to obtain in progenies of general wide hybridization. Taking M16 as an ex-
ample, it was a pollen plant obtained with hexaploid triticale Beagle as mother parent crossed with com-
mon wheat Kedong 5 8 , with FI used in anther culture. Morphologically it is a common wheat, but it is
highly resistant to powdery mildew, with chromosome number of 2n = 44 and high genetic stability. The
chromosome stabilities of mitosis and meiosis were 91 .05 % and 93.23 % , respectively. In situ hy-
bridization and comprehensive identification indicated that it was a 4R addition, 3W3D and 6W6D dou-
ble-substitution line[Io1. M17 and W66 were the pollen plants derived from 6 triticale Beagle x Kedong 58
and Rosner x Kedong 5 8 , respectively. MI7 is resistant to powdery mildew. Comprehensive identification
showed that their chromosome compositions were 1 R and 6R, 1 R and 4R multi-addition lines, respective-
ly. They all possessed 2n = 46 chromosomes. Continued identification through 8 and 11 generations
shows that the chromosome stability varied between 7 4 . 2 % to 85 . 5 % respectively["] . 'Ihese special
types are very important for investigating the recombination and transmission between different alien chro-
mosomes, and further exploiting using useful alien genes. Meanwhile this procedure could directly pro-
duce aneuploids of deletion, telosomics, etc. which were good materials for gene mapping ( see the 3rd
part). ( 2 ) Rapid stability. Since the chromosome-doubled haploids pollen plants became homozygous
diploids, using FI hybrids as materials in anther culture, stable products of chromosome engineering could
be obtained through one generation. This only needs 1 .5 to 2 years. ( 3 ) High efficiency of selection.
Since various types of recombinants and variants could be fully expressed at pollen plant level, in smaller
populations of pollen plants, various types that should be obtained in theory could be selected.
2 Creating alien translocation
Plant geneticists and breeders pay great attention to the investigation of translocation lines, because
it involves study of the chromosome structure and function, exploitation of theoretic genetic problems such
as gene expression and regulation, and transfer of chromosome fragment (gene) into wheat. Meanwhile,
excellent translocation lines could directly be applied to breeding and agriculture.
ears''^' , ~ i e l d m a n ",~~~i a n ~ 'et' ~a ]l . have reviewed the principles and methodologies of inducing
chromosome translocation. These techniques ran be classified into two major groups: ( i ) regulating ph
gene which induces exchange between homoeologous chromosomes to create translocation lines, ( I/ ) in-
ducing chromosome breakage and reunion by irradiation, tissue c ~ l t u r e ~ ~ " and ~ " ' using
~ of gametocidal
genes['s' to create translocation lines
According to our investigation, pollen-clonal variation resulted fmm abnormal meiosis and mitosis.
These two kinds of abnormal cell division could produce chromosome breakage and reunion, which was
the basis of mechanism and theory for obtaining alien translocation lines. The results of our research have
proved that chromosome structure variation could be produced in anther culture, including chromosome

966 Chinese Science Bulletin Vol .44 No. 1 1 June 1999

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breakage and reunion. It was worth noting that Wang et a l . crossed Chinese Spring with l R / l A sub-
stitution line M27 and used the hybrids for anther culture, then directly obtained the WER-1 pollen plant
with two spikes seed-bearing WER- 1-1 and WER- 1-2. Comprehensive identification techniques revealed
that WER- 1-1 was 1R telomere-deleted , and 1A-1 R homozygote translocated[18](Plate 1 ) . Chromosome
composition of 3 generations ( HZ, H3 , & ) remained stable. WER-1-2 was 1R~ telomere-deleted and
I A - ~ Rheter~z~~ote-translocated[~~~
~ . Furthermore, the deletion-translocation gametes could not be pro-
duced by meiotic recombination, it must be produced in male gametophytes (pollen) , indicating that an-
ther culture has supplied an effective pathway for inducing the alien chromosomes' non-Robertsonian
translocation line, which was also suggested by Badaeva et a1 . , who, however, had not got direct ex-
perimental evidence for that. Meanwhile Ren et a l . [''] also suggested the method of inducing the alien
chromosomes' non-Robertsonian translocation line using mono-addition line, but their translocation line
was a product of meiosis which was different from our research design and methodology. In recent years,
we have repeatedly obtained the alien translocation line derived from regenerated pollen plants. For exam-
ple, Hu et a l . [61 and Wang et a l . [231 crossed octoploid tricicale with hexaploid common wheat, then
used septoploid hybrids for anther culture. In HZ, the Robertsonian translocation line (
V209 was identified. zhangl) used the hybrids of Jinghua 3 x M24 (6R/6D substitution line) for anther
culture, and directly obtained the non-Robertsonian translocation line 218. These facts fully indicated
that chromosome engineering of pollen wheat is an efficient way for creating alien translocation line (table
2).
Table 2 Translocation lines of pollen wheat
Pollen plant line Type of translocation Source Character Reference
Non-Robertsonian
DH220-14-2 l R S , lBS-lBL Beagle x Kedong 58 [ 101
1RL telomer double deletion, short straw,
WER-1-1 Chinese Spring x M27 [ 181
1RL-1A homozygous translocation multispikes , big g a i n s
mR-1-2 l R L telomer single deletion,
Chinese Spring x M27 the same as above
lRL-1A heterozygous translocation
1)
Jinghua 3 x M24 resistant to powdery mildew

Robertsonian
resistant to yellow rust,
M O ~ IR~-IB~ Beagle x Kedong 58
leaf strip mosaic, short straw
Spin-1 x Kedong 58 [ 2 3 , 101

DH826- 1 RS-wL Qingzao 180 x M24 resistant to powdery mildew unpublished

DH826-4 RS-wL the same as above the same as above unpublished
DHCl4-1 RS-wL 87(23)2 x M24 the same as above unpublished
DHC14-1 RS-wL the same as above the same as above unpublished

3 Alien gene mapping

Identification of agricultural character-controlling genes in alien chromosomes is very important in
transferring useful genes of alien chromosome into wheat in a planned way. Because rye chromosome al-
most does not pair with homoeologous wheat chromosorne'25-271, it is difficult to .carry out gene (chromo-
some) mapping with conventional method. Taking pollen plants as materials and using morphology, cyto-
genetics, in situ hybridization, and biochemical markers combined with molecular probe method, we

1 ) Zhang, W. J . , Pollen plants chromosome manipulation transfer powdery mildew resistance from 6 R of rye to wheat and genetic re-
search, Doctoral thesis, 1993.

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located some useful genes and markers on 1R and 6R respectively.
( I ) 1 R plant-height gene mapping. Using SDS-PAGE to identify the glutenin composition en-
coded by 1 R and carrying out the backcross reciprocal monosomic analysis, we studied the relationship
between wheat chromosome and rye 1 R chromosome of M27 ( I K/1 D substitution line) . Results indicated
that on 1R chromosomes there were plant height-controlling genes.
( ii ) 1~~awn gene mapping. Awnedness may increase the surface of spike for receiving more
C 0 2 and sun light, thus doubling the photosynthetical activity in the spike. Meanwhile it was proved that
on 1D chromosome of wheat there are awn-inhibitor genes. We crossed Chinese Spring (awnless) with
l R / l D alien substitution line M27 ( a w n e d ) , and used F1 hybrids for anther culture. A set of aneuploids
of pollen plants were obtained, including deletion-translocation line, deletions, telosomic addition and
isoarm addition, etc . They all had a linkage relationship with awnedness . Furthermore, the awnedness-
promoting gene was mapped on the short arm of the 1 R chromosome:I8].
( iii ) 6 ~ resistance
" to powdery mildews gene mapping. Firstly a population of pollen plants re-
sistant to powdery mildew was established. ldentification of resistance to disease and cytology and analysis
of isozyme markers confirmed that 266 is 6 R addition, 267 is 6~~ isoarm addition, 269 is 6~~ telosomic
addition, SO51 is 6 ~ ' deletion
' addition. Meanwhile it has been proved that plants with a long arm of 6 R
are resistant to disease and those without it are susceptible. So the gene resistant to powdery mildew was
mapped on the 6 R long arm chromosonle. I ) Then the 6 R chromosome composition of plants resistant to the
disease was detected by 1 3 DNA probes and 1 isoenzyme marker, and a special band of 6 R chromosome
was detected by one RFLP probe. Therefore, the gene resistant to powdery mildew on 6~~ was accurately
mapped near the probe ps1687[29j, which c:ould be used as gene marker for molecular breeding and for
isolating this gene using molecular map-based cloning method.
4 Perspective
To improve the identification of alien germplasm, our lab and others at home and abroad have estab-
lished and improved comprehensive techniques of identifying morphological characters, resistance to dis-
ease, chromosome banding, in situ hybridization, biochemical markers and molecular probes, etc . Espe-
cially, Jiang and ill:^'] developed plant sequential chromosome banding and in situ hybridization tech-
nique and Zhong et a l . [31'32: developed fluorescence in situ hybridization to pachytene chromosome and
extended DNA fibrils in plants, which has greatly increased the sensitivity and accuracy for identification
of the genetic composition of gene germplasm. All the above-mentioned facts show that the molecular cy-
togenetics in this field is becoming mature.
In recent years, investigation of transgenic plants has been greatly advanced. But a new problem has
arisen. Is chromosome-engineering technique still needed to transfer genes? We and a number of geneti-
cists hold that: First, wheat-related species and genera are a valuable genetic resources bank possessing
great potentialities for wheat improvement, and therefore should be studied and utilized; secondly, wheat
is a polyploid species. Transfemng a gene pool may be more efficient than transferring a single gene, and
chromosome engineering is an efficient method for transferring multi-genes; thirdly, creating alien addi-
tion, substitution and translocation lines is prerequisite for identifying and utilizing of alien genes. Re-
cently, Song et a l . '33i have isolated, cloned and transferred the Xa21 gene which is resistant to white
blight disease of rice by mapping-based cloning technique. Some molecular geneticists considered that
these genes derived from plant itself occupied important positions in plant gene engineering, and there is
no problem of safety. Meanwhile, Tanksley et a1 . 34+35- have paid attention to the application of multi-
gene-controlled quantitative characters such as productivity, good quality and resistance to stress, and
considered that wild germplasm should be exploited. 'l'hey suggested using advanced backcross QTL anal-
ysis. cross method to transfer important genes of quantitative characters, create new germplasm and breed
new. It is the new tendency in the recent development of gene engineering. Alien translocation lines,

1 ) See footnote I ) on page 967

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additiorrsubstitution lines, multi-addition lines, etc . produced by chromosome engineering of pollen
wheat all possess useful alien genes which could be used for investigation and utilization of quantitative
character backcross method. Meanwhile the alleles of dominant and recessive genes of pollen haploids are
codominant, which, similar to the backcross, are able to attain germplasm homozygosity . The close col-
laboration and mutual complementation between techniques of different disciplines are a natural tendency
of science development.

Acknowledgement The authon thank h f . Shao, Q . 0.for his critical reading of this manusrript .

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( Heceiwd December 17 1998 ; accepted February 1 1 , 1999)

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