Advances in agronomy volume 34

ADVANCES IN

AGRONOMY

VOLUME 34


CONTRIBUTORS TO THIS VOLUME
M. J . AMBROSE
R. B . BEVERLY
W. C. GREGORY
UMESHC. GUPTA
Hu HAN
C. L. HEDLEY

W. M. JARRELL
PREMP. JAUHAR
THOMASA . LARUE
JOHN LIPSETT
THOMAS
G. PATTERSON
SHAOQIQUAN
JOSE

G . SALINAS

PEDROA . SANCHEZ
H. K. SRIVASTAVA
J. C. WYNNE

ADVANCES IN

AGRONOMY
Prepared in cooperution with the
AMERICAN
SOCIETY
OF AGRONOMY

VOLUME 34
Edited by N. C . BRADY
Science and Technology Bureau
Agency for International Development
Department of State
Washington, D . C .

ADVISORY BOARD

H. J . GORZ,CHAIRMAN
E. J . KAMPRATH T. M. STARLING

J . B . POWELL J . W. BIGGAR

M . A . TABATABAI
M. STELLY,
EX

OFFICIO,

ASA Headquarters

1981

ACADEMIC PRESS
A Subsidiary of Harcourr Bruce Jovanovich, Publishers

New York

London Toronto Sydney San Francisco


COPYRIGHT @ 1981, BY ACADEMIC PRESS, 1NC.
ALL RIGHTS RESERVED.
NO PART O F THIS PUBLICATION MAY BE REPRODUCED OR
TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC
OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY
INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT
PERMISSION IN WRITING FROM T H E PUBLISHER.

ACADEMIC PRESS,INC.

111 Fifth Avenue, New York. New York 10003

United Kirigdom Edition published b y
ACADEMIC PRESS, INC. ( L O N D O N ) LTD.
24/28 Oval Road, London NWI

7DX

LIBRARY
O F CONGRESS CATALOG CARD NUMBER:50-5598

ISBN 0-12-000734-7
PRINTED IN THE UNITED STATES O F AMERICA
81 82 83 84

9 8 7 6 5 4 3 2 1


CONTENTS
CONTRIBUTORSTO
VOLUME
34 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
PREFACE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ix
xi

ADVANCES IN PLANT CELL AND TISSUE CULTURE IN CHINA

Hu Han and Shao Qiquan
I.
I1.
111.
IV .
V.
VI .

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Anther Culture and Crop Improvement .....................
Some Fundamental Problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Protoplast Isolation, Culture. and Genetic Manipulation . . . . . . . .

Selection of Mutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Miscellaneous: In Vi'itroPropagation through Plant Tissue Culture
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1

2
5
7
9
10
11

HOW MUCH NITROGEN DO LEGUMES FIX?

Thomas A . LaRue and Thomas G . Patterson
I.
II.
I11.
IV .

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Methods of Estimating Fixation by Crops . . . . . . . . . . . . . . . . . . .
Estimates for Major Crops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

15
19
27

34
36

PEANUT BREEDING

J . C . Wynne and W . C . Gregory
I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
II . Germ Plasm Resources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
III . Economic Importance and Breeding Objectives . . . . . . . . . . . . . . .
IV . Breeding and Quantitative Genetics ........................
V . Breeding Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
VI . Interspecific Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
VII . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
V

39
40
44
49
63
65
68
68


vi

CONTENTS

MOLYBDENUM IN SOILS. PLANTS. AND ANIMALS

Umesh C . Gupta and John Lipsett

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I1. Molybdenum Fertilizers. Their Rates and Methods of Application.

13

and Industrial Uses of Molybdenum .....................

75
78
81
85
Responses to Molybdenum on Crops .......................
Factors Affecting the Molybdenum Uptake by Plants . . . . . . . . . . 89
Deficiency and Sufficiency Levels of Molybdenum in Plants . . .
99
Molybdenum Deficiency and Toxicity Symptoms in Plants . . . . . 100
Molybdenum Toxicity and Molybdenum-Copper-Sulfur
Interrelationships in Animals ...........................
105
Summary and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
107
109
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

111. Physiological Role of Molybdenum in Plants . . . . . . . . . . . . . . . .
IV . Determination of Molybdenum in Soils and Plants . . . . . . . . . . . .

V.
VI .
VII .
VIII .
IX .

X.

INTERGENOMIC INTERACTION. HETEROSIS. AND IMPROVEMENT OF CROP
YIELD

H . K . Srivastava
I . Introduction

...........................................

118

I1. Genetics of Mitochondria and Chloroplasts . . . . . . . . . . . . . . . . . . 119
111.
IV.
V.
VI .
VII .

Organelle Involvement in Genetic Phenomena . . . . . . . . . . . . . . .
Genetic Implications of Intergenomic Interactions . . . . . . . . . . . . .
Molecular-Genetic Aspects of Heterosis ....................
Improvement of Crop Yield . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Summary and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

130
147
164
174
182
185

THE DILUTION EFFECT IN PLANT NUTRITION STUDIES

W . M . Jarrell and R . B . Beverly

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I1. System for Expressing Results ............................
111. Mechanisms

IV .
V.
VI .
VII .

...........................................

Treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Dilution Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Concentration Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Practical Implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

197
199
200
202
204
216
219


CONTENTS

VIII . Summary and Future Research Needs ......................
References ............................................

vii

221
222

DESIGNING “LEAFLESS” PLANTS FOR IMPROVING YIELDS OF THE DRIED
PEA CROP

C . L . Hedley and M . J . Ambrose
I.
I1.
I11.
IV .
V.
VI .

VII .

General Introduction .....................................
Comparative Responses of Peas to the Crop Environment . . . . . .
Attaining Maximum Biological Yield per Unit Area . . . . . . . . . . .
Attaining the Maximum Economic Yield per Unit Area . . . . . . . .
Improving the Efficiency of the Pea Fruit . . . . . . . . . . . . . . . . . . .
A Plant Ideotype for Improving Yields of Dried Peas . . . . . . . . .
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

225
229
239
252
265
272
274
275

LOW-INPUT TECHNOLOGY FOR MANAGING OXISOLS AND ULTISOLS IN TROPICAL
AMERICA

Pedro A . Sanchez and Jose G . Salinas
1.
I1.
I11.
IV .
V.
VI .

VII .
VIII .
IX .

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Site Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Selection of Acid-Tolerant Germplasm .....................
Development and Maintenance of Ground Cover . . . . . . . . . . . . .
Management of Soil Acidity ..............................
Phosphorus Management .................................
Management of Low Native Soil Fertility . . . . . . . . . . . . . . . . . . .
Discussion ............................................
Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

280
293
295
308
334
354
380
390
397
398

CYTOGENETICS OF PEARL MILLET

Prem P . Jauhar
I.

I1.
I11.
IV .
V.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Karyotypic Analysis .....................................
Meiosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Abnormal Meiosis and Its Genetics ........................
Haploidy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

408
410
415
417
424


viii

CONTENTS

VI . Polyploidy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Aneuploids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Structural Changes in Chromosomes .......................
B Chromosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Floral Biology and Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . .
Hybridization and Chromosome Relationships . . . . . . . . . . . . . . . .
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

VII .
VIII .
IX .
X.
XI .
XI1 .

428
434
441
446
451
456
472
473

INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

481


CONTRIBUTORS
Numbers in parentheses indicate the pages on which the authors’ contributions begin.

M. J . AMBROSE (225), Department of Applied Genetics, John Innes Institute,
Norwich NR4 7UH, England
R. B . BEVERLY (197), Department of Soil and Environmental Sciences, University of California-Riverside, Riverside, California 92521
W. C. GREGORY (39), Crop Science Department, North Carolina State University, Box 5 / 5 5 , Raleigh, North Carolina 27650
UMESH C. GUPTA (73), Research Branch, Agriculture Canada, P. 0. Box

1210, Charlottetown, Prince Edward Island, Canada CIA 7M8
HU HAN ( I ) , Institute of Genetics, Academia Sinica, Beijing, People’s Republic
of China
C. L. HEDLEY (225), Department of Applied Genetics, John Innes Institute,
Norwich NR4 7UH, England
W. M. JARRELL (197), Department of Soil and Environmental Sciences, University of California-Riverside, Riverside, California 92521
PREM P. JAUHAR* (407), Department of Botany and Plant Sciences, University of California-Riverside, Riverside, California 92521
THOMAS A. LaRUE (15), Boyce Thompson Institute for Plant Research,
Ithaca, New York 14853
JOHN LIPSETT (73), Division of Plant Industry, CSIRO, P. 0. Box 1600,
Canberra City, A . C . S . 2601, Australia
THOMAS G . PATTERSON (15 ) , Boyce Thompson Institute for Plant Research,
Ithaca, New York 14853
SHAO QIQUAN ( I ) , Institute of Genetics, Academia Sinica, Beijing, People’s
Republic of China
JOSE G. SALINAS (279), Tropical Pastures Program, Centro lnternacional de
Agricultura Tropical, Apartado Aereo 67-13, Cali, Colombia
PEDRO A. SANCHEZ (279), Soil Science Department, North Carolina State
University, Raleigh, North Carolina 27650
H. K . SRIVASTAVAT (1 I7), Department of Biology, University of Valle, Cali,
Colombia
J . C. WYNNE (39), Crop Science Department, North Carolina State University,
Box 5155, Raleigh, North Carolina 27650
*Present address: Division of Cytogenetics and Cytology, City of Hope National Medical Center,
Duarte, California 91010.
thesent address: National Agricultural Research Project, Gujarat Agricultural University, Anand
388 1 10 (Gujarat), India.
ix

This Page Intentionally Left Blank


PREFACE
Agronomy is an applied field of endeavor that embodies the combined efforts
of soil and crop scientists to increase the yield and production of field crops. It
utilizes the methods and inputs of the more basic physical and biological sciences
to gain an understanding of plant and soil processes that constrain and/or enhance
crop production. It encourages an integration of the best products of these more
basic sciences into practical crop production systems.
The contributions to this volume illustrate the usefulness of the findings of
agronomists in seeking increased crop yields and production. Four of the articles
deal with genetics and plant breeding. Research on the cytogenetics of an important food crop of the Old World, pearl millet (Pennisetum), is reviewed.
Likewise, the remarkable progress made in recent years in breeding improved
varieties of the common peanut (ground nut) is very effectively covered. Recent
developments relating to improved crop yield through heterosis and intergenomic
interaction are the focus of another article.
During the past 30 years, there has been little interaction between scientists in
the People’s Republic of China and those in the Western world. The contribution
on tissue culture in China discusses an important subject to which the Chinese
have made significant contributions during the past two decades. This method of
crop regeneration and improvement is also beginning to provide plant breeders
with a practical tool that may shorten the time needed to develop new varieties
and may simultaneously increase rates of mutation.
Soil fertility and management are the subjects of four articles. One deals with
factors influencing the amount of nitrogen fixed by legumes, a topic of increasing
importance as rising energy costs force concomitant increases in nitrogen
fertilizer prices. A second related contribution, which will be equally important
to researchers and farmers in large areas of Africa, Latin America, and Asia,
focuses on the management of acid soils of the tropics. Means of alleviating the

constraints placed on resource-limited farmers in these areas are considered.
Soil fertility research methodology is given attention in a contribution concerned with the dilution effect in soil fertility experiments. This review will be
helpful in interpreting data from such experiments. A second soil fertility article
brings us up-to-date on research concerned with molybdenum in soils, plants,
and animals. Knowledge of factors influencing the availability of this element is
helpful in both developing and more developed countries.
The article on the “leafless” pea crop is of special interest to crop
physiologists. It focuses on the comparative responses of ‘‘leafless” and wellleafed peas to environmental and management conditions. The results reviewed
will be of interest to scientists concerned with crop-environment-management
interactions.
xi


xii

PREFACE

As has become the norm for Advances in Agronomy, the subjects covered here
are truly international, and the authors selected to review them come from different parts of the world. We sincerely thank these authors for their contributions, which will be of great value to the world’s crop and soil scientists and,
ultimately, to the farmers on whom we depend to produce our food and fiber.

N. C . BRADY


ADVANCES IN

AGRONOMY

VOLUME 34

This Page Intentionally Left Blank


ADVANCES IN AGRONOMY, VOL. 34

ADVANCES IN PLANT CELL AND
TISSUE CULTURE IN CHINA
Hu Han and Shao Qiquan
Institute of Genetics, Academia Sinica, Beijing, People's Republic of China

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

...................
Wheat
.......................................................
Rice .
..............................................
Corn ..........................
Rubber Tree . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Culture Media . . . . . . . . . . . .

11. Anther Culture and Crop Improvement

111.

IV .

V.
VI .

A.
B.
C.
D.
E.
Some Fundamental Problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. Androgenesis of Gramineous Species in Anther Culture
B. Albinism of Pollen Culture-Derived Plants ................................
C. Genetics and Cytology of Progenies of Regenerated Plants . . . . . . . . . . . . . . . . . . .
Protoplast Isolation, Culture, an
A. Protoplast Isolation . . . . . . .
B. Protoplast Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
C. 'Genetic Manipulation . . . . . . . . . .
Selection of Mutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Miscellaneous: In V i m Propagation through Plant Tissue Culture . . . . . . . . . . . . . . . .
A. Tissue Culture of Drug-Producing Plants.. ................................
B. In Virro Propagation by Plant Tissue Culture . . . . . . . . . . .
References . . . . . . . . . . . . . . .
............................

1

2
2
2
4
4

5

5
5

6
6

I
I
8
8
9
10
10
10
11

1. INTRODUCTION
Plant cell and tissue culture is an old as well as a new area of research in
biological sciences in China. It is old because investigations in this field were
initiated several decades ago. It is new because extensive research has been
carried out and much progress has been made during the last decade, especially
in the last few years.
In the past foreign scientists were not aware of the research work done in
China. As a result of the Sino-Australian Symposium on Plant Tissue Culture
held in May of 1978 in Beijing and the participation of Chinese scientists in
international meetings in recent years, this situation has gradually improved.
1

Copyright 0 1981 by Academic Ress, Inc.
All rights of repduction in any form ~ ~ S E N U I .

ISBN 0-12-ooO734-7


2

HU HAN AND SHAO QIQUAN

II. ANTHER CULTURE AND CROP IMPROVEMENT
During the last ten years, much progress has been made in plant anther culture,
including development of media, standardization of culture conditions, and determination of the response of genotypes to anther culture. In more than a dozen
species, pollen culture-derived plants were first obtained by Chinese scientists
(Table I).
Emphasis has been placed on using anther culture techniques in crop plant
improvement.
A. W H E A T

Plants regenerated from anther culture have been more easily obtained from
materials of hybrid origin than from pure varieties, and significant differences in
plant regeneration frequency have been noted among various hybrid combinations. It is now possible to generate plants through pollen culture of winter and
spring wheat varieties as well as their hybrid combinations. Using materials with
high induction frequency of callus, several dozen green plantlets were regenerated under appropriate conditions from 100 inoculated anthers. Gangsu Academy
of Agricultural Sciences is now capable of producing up to 600 pollen-derived
and colchicine-doubled plants of wheat within a year.
B . RICE

The induction frequency of rice plants regenerated through anther culture has
markedly improved in recent years. For example, Zhang (1980) has been able to
obtain 5106 clumps of green seedlings from 125 combinations of Oryza sutivu
subspecies japonica intervarietal crosses and of indica -japonica intersubspecific
crosses, resulting in thousands of pedigree lines with stable characteristics available for selection and evaluation in 1 year. Thus anther culture can now be used

as a tool in applied plant breeding programs. Up to now several rice varieties
developed through anther culture techniques in rice have been released for commercial production. Hua Yu No. 1, developed cooperatively by the Institute of
Genetics, Academia Sinica and the Institute of Rice Research, Tianjing, is
characterized as high yielding (about 7500 kg/ha), resistant to bacterial leaf
blight, and widely adaptable, and has been grown in suburbs of Tianjing and
Beijing covering an area of thousands of hectares.
Chen Ying et al. (1980b) were able to isolate microspores of rice using a float-


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.


4

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

5

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.

6

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

7

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).

8

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


PLANT CELL AND TISSUE CULTURE IN CHINA

9

successful transfers occurred at a frequency of 2-5% (C. Ma, personal communication).
The characteristics of rape mosaic virus (YMV 15) were studied by introducing
the virus into tobacco (N. tubacum var. Sumsan) mesophyll protoplasts. It was
found that YMVl5 is serologically related to TMV, but the infection pathway is
different. When polyomithine was added, 46-96% of tobacco protoplasts were
infected by YMVI5, and each infected protoplast contained (1-3) X 105 virus
particles (Tian et al., 1977).
Crown gall cell is useful in plant genetic engineering. In general plant tumor
cells induced by different Agrobacterium tumefaciens strains cannot grow on
medium containing o-lactose or D-galactose as the sole carbon source, since
both sugars are toxic to these tumor cells. However, Li Xiang Hui and
Schieder ( 1981) recently discovered that tobacco tumor cells induced by
A . tumefaciens strain B6S3 are different from other tumor cells and can
grow on medium supplemented with D-lactose as the sole carbon source.

This characteristic might provide a selective system for protoplast fusion
and transformation experiments. Somatic hybrids have been obtained through
fusion of protoplasts from B6S3 tobacco tumor cells and those from mesophyll
of N. tubacum cv. Xanthi. The hybrid characteristics were demonstrated by the
presence of octopine identified in the hybrid cells. It now appears possible to
transfer T:DNA segments of the tumor cells to normal ones through protoplast
fusion (X. H. Li, personal communication).

V. SELECTION OF MUTANTS
Cell cultures are also used as means of obtaining variant strains. Investigations
on selection of mutants at plant cell levels got under way recently in China. This
appears to be a promising field of research.
A chlorophyll mutant of rice (HY 101) was obtained that segregated at a
frequency of 19% from an H2strain. The anther callus of variety No. 8126 was
treated with ethyl methane sulfonate (EMS) at an early stage in culture (Hu er
al., 1981) and the aforementioned H 2 strain was obtained from this callus. The
mutant plants are yellowish-green in color and stable in characteristics. The
reciprocal F, s obtained from crosses between the mutant and normal rice plants
were green. The F2population segregated in a 3 : 1 ratio of green yellowish-green
plants. Green and yellowish-green plantlets appeared at a ratio of 1 : 1 in a plant
population obtained from hybrids through anther culture.
Chen et al. (1980a) were able to obtain a BUdR-resistant mutant from soybean cell line SB-1 which was irradiated by 1000-R y rays, and cultured for 20
days on y-rayed B5 medium. It is believed that irradiation might trigger chemical changes in culture medium in addition to the direct effects on the treated cells.


10

HU HAN AND SHAO QIQUAN

Thus the mutation rates may be increased by exposing the calli to the direct and

indirect irradiations.
Ho et al. (1980) were able to select a lysine analog (0L)-resistant tobacco
callus mutant at a low frequency (ca. lo-') by first subculturing tobacco (N.
tubacum L. cv. Gexin No. 5 ) callus through three successive passages on a
medium containing lg/ 1 L-Coxalysine (OL), an inhibitor of callus growth, then
culturing the treated tissue through 6 passages on a medium without the selective
agent OL, and finally treating the callus cultures with 2% EMS. The resultant
mutant is 10 times less sensitive to OL than the parent line, and in general it was
stable through 12 passages of subcultures in the absence of the selective agent. It
was found that the lysine analog-resistant tobacco callus mutant has accumulated
twice the normal level of lysine content, while a different pattern of peroxidase
isoenzyme spectrum has been discovered electrophoretically in the mutant as
compared with that of parent lines.

VI. MISCELLANEOUS: IN VITRO PROPAGATION
THROUGH PLANT TISSUE CULTURE
A number of economically important plants, including seaweeds, have been
propagated through tissue culture in China. In addition, many drug-producing
plants have also been cloned in vitro.
A. TISSUE
CULTURE
OF DRUG-PRODUCING
PLANTS

Peking Pharmaceutical Institute was able to cultivate ginseng callus proliferated from young stems and roots. It was found that the total ginseng saponins was
the same as that of the garden ginseng. For example, the ginseng callus contains
4% (dry weight) saponins, whereas the 6-year-old garden ginseng contained 3%
(dry weight) of this chemical. Zheng and Liang (1976) were able to cultivate the
famous herb Panax noroginseng, and chemical analysis showed that the herb
contained 10.25% (dry weight) of gross saponin and sapogenin, in contrast to the

tuberous roots of the natural plants, which contain 6.06% (dry weight) of the said
drugs only. It has been found that callus tissue of Seopolia ocutagula plant
produced 0.55% (dry weight) of Hyocyamine and seopolamine, whereas fieldgrown plants had only 0.139% of these drugs (Zheng and Liang, 1977).
B. In Vitro PROPAGATION
BY PLANTTISSUE
CULTURE

Since 1975, Wang et al. (1975a,b; Wang and Chang, 1978a,b) have successfully regenerated seedlings from embryo (diploid 2n = 18) and endosperm