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II. Anther Culture and Crop Improvement

II. Anther Culture and Crop Improvement

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PLANT CELL AND TISSUE CULTURE IN CHINA



3



Table I

Species in Which Regenerated Plant Was First Obtained in China

Species



Year"



References



Wheat

(Triticum aesrivum)



1971



Triticale

(Triticale)

Wheat-wheat grass hybrid

(Triticum aestivum X

Agropyron glaucum)

Maize

(Zea mays L.)

Boom corn

(Sorghum vulgare)

soybean

(Glycine max)

Alfalfa

(Medicago denticulata)

Rubber tree

(Hevea brasiliensis)

Poplar

(Populus nigra L.)

Pepper

(Capsicum annum L.)

Brassica pekinesis

Chinese cabbage

(Brassica chinesis)

Sugar beet ( 2 n =4x=36)

(Beta vulgaris L.)



1971



Ouyang et al. (1973)

Wang et al. (1973)

Chu ef al. (1973)

Sun et al. (1973)



1973



Wang et al. (1975a,b)



1975



Ku et al. (1975)



1978



Zhao (1978)



1980



Yin et al. (1980)



1979



Xu (1 979)



1977



Chen et al. (1978)



1974



Wang et al. (1975a,b)



1971



Wang et al. (1973)



1973

1977



Teng and Kuo (1977)

Chung et al. (1977)



1973



1979



Breeding Lab. Heilingchiang Sugarbeet

Institute (1973)

Shao (1979)



1979



Chen et al. (1980~)



I979



Sun (1979)



1978



He Ze Chinese Medicine Laboratory

( 1978)



Sugar beet (2n=2x=18)

(Beta vulgare)

Sweet orange

(Citrus microcarpa)

Flax

(Linum usitatissimum)

Rehmannia glurinoso



"Indicates the year the plants were obtained.



ing culture method-subjected pollen grains liberating continuously from dehiscing anthers-and obtained many calli and green plantlets. More recently green

seedlings were directly induced from pollen grains on media without phytohormones.



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HU HAN AND SHAO QIQUAN



C. CORN



It is valuable and beneficial to develop corn inbred lines through anther culture. Much investment of effort has gone into anther culture investigations of

corn inbred lines in China. Different starting materials have been used, resulting

in 25 inbred lines obtained by 14 institutes and universities in the past 2 years.

Meanwhile a test crossing of some inbred lines is under way.

Uniform canopy structure was demonstrated in these inbred lines. The comparison of corn inbred lines and the parent varieties “525” and “Gui No. 622”

for plant height, ear length, 100 grain seed weight, and seed weight per ear

revealed that the coefficient of variation was much lower in the pollen-derived

inbred lines (Chen et al., 1979a).

For example, a corn inbred line, Qunhua No. 105, has recently been produced

from hybrid corn Qundan No. 105 through anther culture (Wu er al., 1980).

Results of the test cross with this line showed that of the ten hybrid combinations

tested, nine (90%) were superior than the control. Therefore, Qunhua No. 105 is

a promising corn inbred line.

Using corn varieties“Ba Tang Bai” and “Qundan No. 105” as parental

materials, Cao (personal communication) and Wu and Zhong (1980) have been

able to obtain maize cell clones through anther culture techniques in recent years.

Gu (personal communication) and Cao (personal communication) have subcultured the cell clones derived from “Ba Tang Bai” every 4 weeks, and now

these clones have gone through 30 generations of subculturing. The clones can be

grouped into four types according to the degree of their totipotency. The No. 1

clone has intensive capacity of differentiation and regeneration, and green

plantlets have been readily regenerated from it. Cytological studies of 427 cells

of No. 1 clone and of 645 root tip cells of 35 plants regenerated from No. 1 clone

revealed that 89.7% of the former and 87.4% of the latter were haploids. This

type of cell clone, which has such high regeneration capacity and genetic stability, is a rare material in plant tissue culture.

D. RUBBERTREE



Plants of rubber tree (Hevea brasiliensis) were regenerated in 1977 from

anthers cultured in vitro (Chen, 1978). In the last 2 years Chen et al. (1979b) have

studied the relationship between callusing anthers and the formation of pollen

embryoids of Hevea brasiliensis. They found that after 20-30 days of culture

80% cells at metaphase of mitotic division were observed to be diploids (2n =

36), while after 50 days of inoculation only 10% were diploids and 69% were

haploids. The result shows that after 50 days of culture the callus derived from



PLANT CELL AND TISSUE CULTURE IN CHINA



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anther walls was degenerating, whereas the haploid tissue of microspore derivation was dividing vigorously. In order to obtain haploid embryoids or calli it is

appropriate to transfer callusing anthers to differentiation medium after 50 days

of inoculation.

A total of 238 cells from 46 embryoids were observed at mitotic metaphase. Of

these, 4% had 9 chromosomes, 80% had 18 chromosomes, 15% had 27 chromosomes, and 1% had 32, 36, or 45 chromosomes. The preponderance of haploids

originating from embryoids was demonstrated. Root tips of 18 plantlets were also

analyzed cytogenetically and similar results were obtained (Chen et a f . , 1979b).

E. CULTURE

MEDIA



Several culture media developed first by Chinese scientists are now widely

used within the country as well as abroad. For example, the potato medium (First

Group, 1976; Ouyang e t a l . , 1977; Chuang e t a l . , 1978), which contains 10-30%

potato extracts, works as well or even better than the Miller’s and MS’s media.

The use of potato extract as a medium component has greatly improved callus

induction.

N6 is another efficient medium developed by Chu et al. (1975) in which

ammonium salts in optimum concentration are added along with nitrate salts. The

frequency of anther callus induction on N6 medium is higher than that on Miller’s and MS media. It was as high as 16% (even 50% in some cases) for

intervarietal hybrids of rice.



Ill. SOME FUNDAMENTAL PROBLEMS

A. ANDROGENESIS

OF GRAMINEOUS

SPECIES IN ANTHER

CULTURE



The processes of androgenesis during anther culture, particularly of gramineous species such as wheat, triticale, rye, barley, rice, and corn, were studied in

the last few years. In addition to A and B pathways, others (such as C, D, and E

pathways) were discovered (Sun, 1978). It is obvious that the abnormal

mechanisms occur in early stages of pollen grain development during anther

culture of wheat under different incubation conditions, particularly at low temperature (Tseng and Ouyang, 1980). Results obtained are useful in establishing a

scientific basis not only for improving the frequency of callus induction and plant

regeneration, but also for the elucidation of the causes of spontaneous chromosome doubling and chromosome aberrations in some plants, as well as for understanding the mechanisms of microspore differentiation.



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HU HAN AND SHAO QIQUAN



B. ALBINISM

OF POLLEN

CULTURE-DERIVED

PLANTS



In gramineous species, more than one-third of the plantlets regenerated from

anther culture are albinos. For the effective use of anther culture in haploid

breeding it is important to discover the causes of albinism and seek remedies to

overcome this problem. Systematic studies of this problem should also contribute

to the understanding of the genetics, physiology, and cytology of chloroplast

development.

Systematic investigations on the occurrence of albino plantlets in rice revealed

that the viability of the developing plastid in chloroplast was the direct cause of

albinism. A barrier to the nucleic acid translation system of the proteins needed

for the development of a plastic lamellar system (a barrier to gene expression) is

perhaps present (Wang et al., 1978; Liang et al., 1978). Recently components of

soluble protein and ribosomal RNA from green and albino pollen culture-derived

plantlets of rice have been analyzed by polyacrylamide gel electrophoresis. It

was found that little or no band 3 (Fraction I protein), 23 S RNA, and 16 S RNA

are present in albino plantlets (Sun et al., 1978). Together with the evidence

obtained from other investigations, it was suggested that the albinism is caused

by the impairment function of DNA.



c. GENETICSA N D CYTOLOGY OF PROGENIES OF REGENERATED

PLANTS

1 . Genetic Stability



Genetic stability of plants derived from tissue and anther culture has been

investigated by numerous scientists in China as well as abroad. These investigations revealed that the progenies of tissue and anther culture-derived plants of

crop species are highly stable. Anther and tissue culture-derived progenies of

rice, tobacco, and wheat were compared with their parental varieties by several

research groups. The coefficient of variation (CV) values for several agronomically important traits were similar in two groups or sometimes small in pollen

culture-derived progenies. Ninety percent of the diploid lines obtained from

doubled haploids of anther culture derivation were homozygous, whereas only

10% showed segregation (Hu et al., 1980).

Cytological examination of root tip chromosome number of 54 H I (regenerated) plants of wheat showed that about 90% of the regenerated plants were either

haploids or homozygous diploids. The analysis of PMCs of 72 H I wheat plants

(Hu et al., 1980) showed that 87.5% of these regenerated plants were either 3x or

6x. Similar results were obtained from examination of somatic chromosome

numbers of field-grown, pollen culture-derived plants of wheat. Over 80% were



PLANT CELL AND TISSUE CULTURE IN CHINA



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either haploids (3x) or homozygous diploids (6x). These observations favor the

use of the anther culture technique in applied plant breeding programs.



2 . Variation



,



Cytological investigations of H plants revealed that approximately 10% have

deviant chromosome numbers, such as monosomics, nullisomics, aneupolyhaploids, pentaploids, and octoploids. These plants with variant chromosome numbers have proved useful in chromosome and genome engineering. The

mechanisms underlying the origin of these variant plants have been discussed by

D’Amato (1978).

Variability of chromosome number of regenerated wheat plants has also been

analyzed in our lab. Mixoploids and cells showing tripolar mitosis, which could

result in heteroploids and aneuploids, were observed in wheat root-tip and calk

dicentric chromosomes and chromosome fragments in pollen calli, suggesting

that in development of anthers cultured in vitro, chromosomevariation may occur

(Hu et al., 1980). Moreover, in early stages of pollen callus formation in virro,

nuclear asynchronous divisions, fusion of different nuclei, and endomitosis were

occasionally observed. These abnormalities in mitosis may lead to the variation

in the chromosome number of plants regenerated from anther culture.

From the foregoing, we consider that either the stability or the variability of

the chromosome numbers of progenies of plants derived through anther culture is

useful in crop improvement as well as in genetic studies.



IV. PROTOPLAST ISOLATION, CULTURE, AND

GENETIC MANIPULATION

A. PROTOPLAST

ISOLATION



Studies on protoplast isolation were initiated in China in 1973. Mesophyll cells

and cells from cultured calli, especially of cereal crops, have been often used. In

order to overcome the difficulty of inducing differentiation in long-term subcultures, stem tips of hybrid rice seedlings were cut and incubated, and spherical

protoplasts were then released (Guangdong Institute of Botany, 1978). Potrykus

et al. (1977) also reported that differentiation is more easily induced in corn

protoplasts obtained in this manner than those from calli cultured for a long time.

A complex enzyme extracted from Trichoderma viride EA3-867used in protoplast isolation has been as effective as cellulase Onozuka R-10made in Japan

(Hsu, 1978).



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HU HAN AND SHAO QIQUAN



B. PROTOPLAST

CULTURE



Plants have been regenerated successfully from protoplasts of tobacco, petunia,

dnd carrot (Li et al., 1978a,b; Wu et al., 1977). Efforts to generate plants from

protoplasts derived from cereals, legumes, and other species have been less

successful (Yen and Li, 1979; Li et al., 1978a,b; Cytology Lab., 1977; Tsai et

al., 1978).

Recently a two-layer culture method, i.e., liquid in the upper layer and solids

in the lower, has been developed, and plants have been regenerated from

mesophyll protoplasts of Nicotiana rustica x N . alata (Hsia et al., 1979). Moreover, typical division of cells regenerated from wheat and barley mesophyll

protoplasts has been observed at about 0.1% frequency (Li, 1979a,b).

Of the nutrients explored, vitamin and other organic compounds are very

important to protoplast division, while glucose and low levels of other sugars

have favorable effects on wheat protoplast culture. When protoplasts were cultured under lower osmotic conditions, e.g., 0.4 M glucose, budding, bulbing,

and anuclear subprotoplasts were generated probably due to rapid swelling of

protoplasts and incomplete cell wall formation (502 Group, 1974).

More recently somatic hybrid plants have been regenerated through fusion of

protoplasts from somatic cells of N . tabacum and those of N . rustica. Examination of chromosome number and identification of peroxidase isoenzymes revealed that the plants are of somatic hybrid origin (Wang et al., 1981; Gong

et al., personal communication).

C . GENETICMANIPULATION



Homologous fusion of mesophyll protoplasts of wheat, corn, and other species,

and heterologous fusion of those of wheat and Viciafaba by adding NaN03 to

culture medium were first obtained at a low frequency of approximately 4%

(Sun, personal communication). Using Kao’s fusion technique with high pH,

calcium ion solution, and PEG,interspecific fusion was induced between protoplasts from wheat yellowing leaves and those from green leaves of petunia. The

fusion occurred at a frequency of 25%, and 10% of the fusion bodies were of

heterologous origin. Nuclear staining confirmed that the fusion bodies were

heterokaryons. Some of the fused protoplasts regenerated cell wall, underwent

cell division, and developed into small calli after being transferred to fresh

medium, but they could not be identified as hybrids due to lack of a selection

system.

In chloroplast transplantation research, chloroplasts of wheat and spinach were

introduced into carrot callus protoplasts with PEG used as inducing agent and



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