genetics of callus formation and plant regeneration in
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LSU Historical Dissertations and Theses Graduate School
1988
Genetics of Callus Formation and Plant Regeneration in Rice Genetics of Callus Formation and Plant Regeneration in Rice
(Oryza Sativa L.). (Oryza Sativa L.).
Qi Ren Chu Louisiana State University and Agricultural & Mechanical College
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G enetics o f callus form ation and plant regeneration in rice ( Oryza sativa L .)
Chu, Qi Ren, Ph.D.
The Louisiana State University and Agricultural and Mechanical Col., 1988
UMI300 N. Zeeb Rd.Ann Arbor, MI 48106
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UMI
GENETICS OF CALLUS FORMATION AND PLANT REGENERATION IN RICE (ORYZA SATIVA L.)
A Dissertation
Submitted to the Graduate Faculty of the Louisiana State University and
Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of
Doctor of Philosophy
in
The Department of Agronomy
byQi Ren Chu
B.S., Shanghai Normal University, China, 1977 M.S., University of The Philippines at Los Banos, 1982
May 1988
ACKNOWLEDGMENT
Sincere thanks are conveyed to the author's major
professor, Dr. Timothy P. Croughan, for providing financial
support, assistance in structuring the research project,
advice on academic course work, arranging for the use of
equipment and facilities, guidance on scientific exploration,
encouragement to overcome various difficulties, helpful
criticism on experiments and manuscripts, and for his sincere
concern for my personal welfare, all of which contributed to
the successful completion of this program.
Great appreciation is extended to Dr. Suzan S. Croughan
for her valuable instructions on various techniques involved
in microscope photography, for the fruitful suggestions on
SAS data analysis, and for the useful suggestions regarding
the preparation of this manuscript.
The author is indebted to Dr. Edward P. Dunigan, Head of
the Department of Agronomy, for his helpful guidance on
academic and scholastic matters. Special thanks are also
expressed to the other members of the advisory committee: Dr.
William J. Blackmon for his insightful suggestion to make
biotechnology an important coursework focus; Dr. Freddie A.
Martin for his important suggestions on experimental design;
and Dr. Elaine M. Nowick and Dr. Kent S. McKenzie for their
review of the thesis proposal and proposed coursework.
Special thanks are expressed to Dr. Joseph A. Musick,
ii
Resident Director of the Rice Research Station, for his
encouragement and the provision of facilities and field
research area for my doctoral research.
The author is grateful to Mona M. Meche, H. J. Huang,
Dannell A. Trumps, and the team of student workers for their
assistance with the laboratory and field experiments.
The doctoral program could never have been possible
without the strong support of the author's government,
colleagues, and family. The Shanghai Academy of Agricultural
Sciences and the Shanghai Municipal Government provided the
official approval required; his colleagues in Shanghai
provided continuity in the author's research program during
his absence; and his father, Professor Chu Qi, until he
recently passed away, provided continual spiritual
encouragement.
Finally, gratitude is wholeheartedly expressed to his
wife, Xin Hua Wang, for her understanding, patience,
dedication, assistance with the research, and personal
sacrifices, without which the attainment of this degree would
not have been possible.
iii
TABLE OF CONTENTS
Page
ACKNOWLEDGMENT ............................................ ii
LIST OF TABLES .......................................... viii
ABSTRACT .................................................. xi
INTRODUCTION .............................................. 1
LITERATURE REVIEW ........................................ A
I. HISTORY OF RICE TISSUE CULTURE.... ................. 5
1.1. Culture of diploid tissue .................... 5
1.2. Culture of haploid tissue .................... 6
1.3. Cell suspension culture ...................... 7
1.4. Rice protoplast culture ...................... 9
II. FACTORS AFFECTING RICE TISSUE CULTURE ........... 11
II. 1. Culture media ........ 11
11.2. Genotype ....................................... 14
11.3. Culture conditions and pretreatments ...... 17
III. SOMACLONAL VARIATION IN RICE ................... 19
111.1. Somaclonal variation in diploid culture .. 19
111.2. Somaclonal variation in haploid culture .. 21
111.3. Somaclonal variation in
biochemical traits .......................... 23
IV. VARIETAL IMPROVEMENT THROUGH
RICE TISSUE CULTURE .............................. 25
IV.1. Use of anther culture in rice breeding .... 25
IV.2. Use of somaclonal variability in rice
iv
breeding 26
Chapter
I. GENETICS OF PLANT REGENERATION IN IMMATURE PANICLE
CULTURE OF RICE (ORYZA SATIVA L.) 27
Abstract ........................................... 27
Introduction ........................................ 28
Materials and Methods ............................ 29
Results and Discussion ........................... 32
1. Genotypic differences in plant regeneration
from immature panicle culture ............... 32
2. Plant regeneration rates in
a 4 x 4 diallel cross and
the BCF^, F 2 > and F 3 progeny ................ 33
3. Estimates of gene effects .................. 34
4. Heritability estimates ...................... 36
References ........................................ 37
II. GENETICS OF CALLUS FORMATION IN ANTHER CULTURE
OF RICE (ORYZA SATIVA L.) 46
Abstract .......................................... 46
Introduction ...................................... 47
Materials and Methods ........................... 49
Results and Discussion .......................... 52
1. Genotypic differences of callus formation
rates in ten rice varieties ................ 52
v
2. Callus formation rate in a 4 x 4
diallel cross ................................ 52
3. Callus formation rate in BCFj, F2 > and
F 3 progeny ................................... 53
4. Generation means and estimates of
gene effects ................................. 55
References ......................................... 58
III. GENETICS OF PLANT REGENERATION IN ANTHER
CULTURE OF RICE (ORYZA SATIVA L.) 69
Abstract ........................................... 69
Introduction ....................................... 70
Materials and Methods ............................ 72
Results and Discussion ........................... 74
1. Green and albino plant regeneration rates
for parents and F^'s ......................... 74
2. Green and albino plant regeneration rates
in F2 and F3 populations ...................... 75
3. Green and albino plant regeneration rates
in 24 BCF1 's .................................. 76
4. Genetic estimates of the green plant and albino
regeneration rates in 7 generations .......... 77
References ......................................... 79
SUMMARY ................................................... 91
BIBLIOGRAPHY 94
APPENDICES
1. Varietal improvement through anther culture
and tissue culture ................................... 117
2. Generation mean analysis: model and formula ...... 122
VITA ............. 125
vii
LIST OF TABLES
Chapter I. GENETICS OF PLANT REGENERATION IN IMMATURE
PANICLE CULTURE OF RICE (ORYZA SATIVA L.)
Table Page
1. Genotypic differences in plant regeneration from
immature panicle culture of 1 0 rice varieties ....... 40
2. Plant regeneration in a 4 x 4 diallel cross ........ 41
3. Plant regeneration from immature panicle
culture of F 2 and F3 populations ..................... 42
4. Plant regeneration from immature panicle
culture of 24 backcrosses ............................. 43
5. Generation means for plant regeneration
from immature panicle culture of rice ............ 44
6 . Mean estimates of six gene effects
in plant regeneration ................................. 45
Chapter II. GENETICS OF CALLUS FORMATION IN ANTHER
CULTURE OF RICE (ORYZA SATIVA L.)
Table Page
1. Callus formation rates of 10 varieties ............... 62
2. Callus formation rates of parents and their F^'s .... 63
3. Callus formation rates of F2 generation .............. 64
4. Callus formation rates of F 3 generation .............. 65
viii
5. Callus formation rates of 24 BCF^'s .................. 6 6
6 . Generation means of callus formation rates
in anther culture of rice .............................. 67
7. Estimates of the genetic components of generation
means for callus formation rates of 1 2 crosses ....... 6 8
Chapter III. GENETICS OF PLANT REGENERATION IN ANTHER
CULTURE OF RICE (ORYZA SATIVA L.)
Table Page
1. Plant regeneration in four parents and
their 12 F ^ 1 s .......... <■.............................. 83
2. Green and albino plant regeneration rates
in F 2 .......... 84
3. Plant regeneration in F 3 generation ................. 85
4. Regeneration rates of 24 BCF^'s ..................... 8 6
5. Mean plant regeneration rates for parents
and progeny ............................................. 87
6 . Genetic components of plant regeneration rates
of parents and progeny ...... 8 8
7. Mean albino plant regeneration rates
for parents and progeny .................. 89
8 . Genetic components of albino regeneration rates ..... 90
ix
Appendix 1
Table Page
1. A 4 x 4 diallel cross made in 1986 118
2. Backcrosses made in summer, 1986 .................... 119
3. Plant regeneration from anther culture
and immature panicle culture ......................... 1 2 0
4. Regenerated lines for field evaluation
and selection in 1987 121
Appendix 2
Table Page
1. Coefficients for genetic components of
means of each of seven generations .................. 1 2 2
2. Equations for calculating six parameters ........... 123
3. Equations for calculating variances
of estimates ........................................... 124
x
ABSTRACT
Callus formation and plant regeneration rates for both
immature panicle and anther culture of rice were studied in
several populations, including parents, F^, F 2 , F 3 , and
backcrossed generations. Significant genotypic differences
in regeneration and callus formation rates were observed. In
immature panicle culture, F^'s generally produced rates of
regeneration close to their high parents. Generation mean
analysis of regeneration rates revealed the importance of
dominance effects (d) and epistatic effects (aa, ad, and dd)
in immature panicle culture. In contrast, production of
callus by 1 2 Fj hybrids in anther culture was closely related
to the low parents, suggesting that suppression of callus
formation was dominant. Generation mean analysis revealed
significant contributions from dominance (d) and epistatic
effects to the trait. Plant regeneration rates in anther
culture appear heritable. The mean regeneration rates in
F^'s showed overdominance and recessive characters in some
crosses and significant reciprocal differences were found for
Lemont/Short Tetep.
A total of 6,332 immature panicles and 375,873 anthers
were cultured, producing 28,395 immature panicle derived
regenerates and 15,003 anther derived regenerates.
Evaluation of these materials has identified germplasm
potentially useful for rice varietal improvement.
xi
INTRODUCTION
A significant portion of worldwide agricultural research
focuses on rice. Progress from this research has included
the development of modern rice cultivars and advancements in
tissue culture technology. Integration of conventional
breeding approaches with tissue culture techniques offers new
potentials for varietal improvement. In recent years, rice
anther culture breeding procedures have been established and
used to develop over 1 0 0 new cultivars and superior breeding
lines (Zhang and Chu, 1986; Loo and Shu, 1986; Chen, 1986a,
b).
Rice breeding depends on the efficient production of
genetic variability and genetic recombination coupled with
the evaluation and selection of superior genotypes. Tissue
culture complements this conventional approach through the
contribution of germplasm derived from diploid and haploid
culture m vitro. Plant regeneration rates are a critical
factor influencing the potential contribution of tissue
culture techniques to breeding. Much of the early research
on tissue culture techniques was directed towards improving
culture media, determining the best culture environment, and
exploring various pre- and postculture treatments for
explants and regenerating plantlets (Chen, 1986a). Even
though a significant increase in plant regeneration rates has
been obtained in anther culture of japonica hybrids in the
2
last 1 0 years, low regeneration rates in indica rice remains
a problem (Zhang and Chu, 1986).
Genotypic differences in callus formation and plant
regeneration have been reported since the first successes in
haploid culture of rice (Niizeki and Oono, 1968). These
differences exist among Oryzae species, and also within the
indica and japonica subspecies of cultivated rice (Mukherjee,
1973; Chen et al., 1974; Oono, 1975; Chen and Lin, 1976; Yin
et al., 1976; Woo and Huang, 1980; Wakasa, 1982; Abe and
Sasahara, 1982; Sheng et al., 1982; Ding et al., 1983; Xui
and Liu, 1984; Zhang and Chu, 1985; Miah et al., 1985; Chu et
al., 1986; Boyajiev and Kuong, 1986; Davoyan, 1987). The
capacity for plant regeneration appears to vary considerably,
and the genetic basis for this trait is not well understood.
Indica varieties in general have low plant regeneration
rates, typically less than 1 %, which severely limits the
efficiency of anther culture of hybrids entailing indica
parents. Regeneration rates for japonica types are somewhat
better, but still low. Screening germplasm with higher rates
of plant regeneration, and understanding the basis of that
improved response, is therefore important for using anther
culture techniques in breeding.
The present studies entail an evaluation of the
culturability of ten rice varieties and the establishment of
a complete 4 x 4 diallel cross from selected parents. The
objectives of the studies were to elucidate genotypic
differences in culturability among a broad genetic range of
3
indica rice varieties and to investigate the genetic
background of the trait in both immature panicle culture and
anther culture of rice.
LITERATURE REVIEW
The first reports of rice tissue culture emerged from
Japan in the early 1950's and described the successful
culture of roots from rice seedlings (Fujiwara and Ojima,
195A). In the next few years, successes were also reported
for rice embryo culture, shoot meristem culture, stem node
culture and cell suspension culture (Amemiya et al., 1956a,
b; Kawata and Ishihara, 1957; Nakajima and Morishima, 1958;
Furuhashi and Yatazawa, 1964; Maeda, 1965). Studies in the
mid 1960's reported an apparent requirement for the inclusion
of auxin and yeast extract in the culture medium to induce
callus formation and growth (Yamada et al., 1967a, b;
Yatazawa et al., 1967). However, plant regeneration from in
vitro culture remained to be achieved.
Several significant advances in achieving regeneration
from rice were reported in 1968. Plants were successfully
regenerated from cultured cells derived from embryos (Tamura,
1968), roots (Kawata and Ishihara, 1968; Nishi et al., 1968),
seeds (Maeda, 1968), and anthers (Niizeki and Onoo, 1968).
Since then, rice plants have been regenerated from cells
derived from a wide range of explants including shoots,
roots, leaf blades, leaf sheaths, unpollinated and pollinated
ovaries, immature and mature endosperm, mesocotyl, seedlings,
immature panicles, seeds, anthers, and pollen grains (Yamada
and Loh, 1983).
5
I. HISTORY OF RICE TISSUE CULTURE
I. 1. Culture of Diploid Tissue
The first reports of regeneration in rice involved the
culture of rice diploid tissue. Kawata and Ishihara (1968)
succeeded in regenerating plants from callus derived from
root tips. The medium used for callus induction contained
various concentration of the auxins 2,4-dichloro-
phenoxyacetic acid (2,4-D) and indoleacetic acid (IAA). The
calli induced were transferred to differentiation medium
containing 1% casein hydrolysate, 5.7 um IAA and no
cytokinins. The regeneration rate was very low and plantlet
growth was poor. Nishi et al. (1968) induced callus
formation from rice roots on LS medium (Linsmaier and Skoog,
1965) containing 9 um 2,4-D. Plant regeneration was obtained
when the callus was transferred to similar medium without
2,4-D and incubated in the light. Tamura (1968) reported the
formation of shoots through organogenesis from embryo-derived
callus and indicated that the leaf primodia originated within
minute indentations of the surface on the callus. Nishi and
Mitsuoka (1969) reported plant regeneration from callus
derived from various parts of the rice plant. Diploid plants
were recovered from callus induced from both embryo and shoot
node explants. Wu and Li (1971) reported the regeneration of
plants from cotyledonary nodes of rice. Nishi et al. (1973)
recovered plants from callus on LS medium containing 2,4-D
but lacking cytokinins. Mascarenhas et al. (1975a, b)
achieved plant regeneration from callus initiated from
6
seedling mesocotyl segments. Immature endosperms harvested 3
to 7 days after pollination and inoculated on culture media
produced callus capable of regenerating plants (Nakano et
al., 1975). Henke et al. (1978) regenerated plants using
scutellar tissue as the explant. Similar success at callus
induction and plant regeneration has been reported using
mature endosperm (Bajaj et al., 1980). Regeneration has also
been reported from leaf sheath and leaf blade culture
(Bhattacharya and Sen, 1980; Yan and Zhao, 1982).
Regeneration from immature panicle culture of rice was
first reported by Tang (1979) and has been followed by
several reports citing genetic variability produced through
immature panicle culture (Zhao et al. 1982; Ling et al.,
1983a, b; Zhang and Chu, 1984; Croughan et al., 1985, 1986;
Chen et al., 1985).
I. 2. Culture of Haploid Tissue
Niizeki and Onoo (1968) were the first to regenerate
plants from rice anther culture. Anthers containing immature
pollen were inoculated onto callus induction medium
containing 9.4 tun naphthaleneacetic acid (NAA), 4.9 um 2,4-D,
10 tun kinetin (KT), and 3 ppm yeast extract. Plant
regeneration was achieved when callus was transferred onto
the same medium lacking 2,4-D. Nishi and Mitsuoka (1969)
successfully obtained regeneration from both anther and ovary
culture. Successful anther culture of indica/japonica
hybrids was reported by Woo and Tung in 1972.
7
Because of the potential for contributing to rice
varietal improvement, rice anther culture programs were soon
initiated by several research groups in China (Inst. Genet.
Acad. Sin., 1972; Inst. Bot. Acad. Sin., 1972; Chen et al.,
197A; Res. Group of Rice, Shanghai Acad. Agric. Sci. 1976;
Chen and Li, 1978; Hu, 1978; Loo, 1979, 1982; Chu, 1982a, b;
Sheng et al., 1982; Woo and Chen, 1982; Zhang, 1982; Zhang
and Chu, 1986; Loo and Shu, 1986). Considerable subsequent
work has been directed towards improving culture media and
techniques which enhanced the production of callus and
plantlets. Important findings from research conducted around
the world have included improvements in culture media,
identification of the best maturity stage of pollen to use,
and the identification of cold pre-treatments as beneficial
in improving callus formation rates (Rush et al., 1982;
Yamada, 1982; Chu, 1982b; Chaleff and Stolarz, 1982; Chung,
1982; Woo and Chen, 1982; Beauville, 1982; Bajaj, 1982).
1.3. Cell Suspension Culture
Maeda (1965) obtained cell suspension cultures by
transferring calli into liquid medium on a rotary shaker. By
continuous shaking at 1 0 0 rpm, cells and small aggregates
were separated from the callus and continued growth in the
liquid medium. Ohira et al. (1973) conducted an extensive
study of the nutrition of rice cell suspension cultures,
including the role of auxin in the culture media. They found
that amino acids were essential for suspension culture. This
8
suspension culture system was modified by Ye (1984) and used
to select salt tolerant cell lines. Single cells and 1 to 3
cell aggregates were isolated from suspension using a metal
mesh with 30 micron pores. The nutrient medium was a
modified B5 medium (Gamborg et al., 1968) containing 9 um
2,4-D and plant regeneration was obtained on MS medium
(Murashige and Skoog, 1962) with an elevated sucrose
concentration.
Kowyama and Shibata (1985) studied the radiation
sensitivity of various genotypes in cell suspension culture
through subjecting 3 indica and 3 japonica varieties to 2.5 -
15 KR doses of X-rays. They concluded that sensitivity was
under polygenic control. Flowers et al. (1985) investigated
the effects of sodium chloride on cell suspension cultures of
rice, and regenerated plants tolerant to 2% NaCl. By using
root-tip derived suspension cultures of Taipei 309, Zimmy and
Lorz (1986) obtained plant regeneration via organogenesis.
Cell cultures were established in liquid MS medium containing
9 um 2,4-D and shoot and root differentiation occurred on
medium lacking auxin but containing kinetin. Zhou et al.
(1986) increased the inositol content 4 fold in liquid MS
medium and obtained plant regeneration from single cell
culture.
1.4. Rice Protoplast Culture
The enzymatic isolation of rice protoplast from leaves,
roots and callus was reported by Maeda and Hagiwara (1974),
9
Liu (1975), and Tseng et al. (1975). Deka and Sen (1976)
obtained high yields of isolated protoplasts from leaf sheath
and stem derived callus. In this case, callus was treated
with an enzyme solution consisting of 2% pectinase and 3%
cellulase. They used 0.45 M mannitol as the osmoticum and
resynthesis of cell walls occurred after 24 hours. However,
no plant regeneration was reported from their experiments.
Although several papers have been published on the isolation
of rice protoplasts (Anon., Peking Inst. Bot., 1975; Lai and
Liu, .1976; Cai et al., 1978; Niizeki and Kita, 1981), low
frequencies for protoplast division and failure to regenerate
plants remained a problem.
Fujimura et al. (1985) were the first to report actual
regeneration of rice plants from protoplast derived callus.
Protoplasts were derived from calli initiated from the
scutellum of rice embryos, and isolated with an enzyme
solution containing 1% Macerozyme RIO, 4% cellulase RS, 0.5%
calcium chloride, 0.5% potassium dextran sulfate and 0.4%
mannitol. After 3 hours of enzyme treatment, isolated
protoplasts were suspended on R2 medium (Ohira, 1972) with 9
um 2,4-D and B5 vitamins. The next researchers to report
success with plant regeneration from protoplasts were Yamada
et al. (1986). They attributed the success to the choice of
donor genotype, the selection of a cell line with embryogenic
capacity, and the development of an improved medium, RY-2.
By using Taipei 309, researchers in Dr. Cocking's laboratory
in England reported a reproducible system of protoplast
10
culture and plant regeneration from rice protoplasts
(Abdullah et al., 1986; Thompson et al., 1986, 1987). The
principle factors contributing to the success in Cocking1s
program were the choice of donor genotype, establishment of a
viable cell suspension system, high yields of isolated
protoplasts following enzymatic treatment, the use of KPR
medium for protoplast culture (Kao et al., 1975), and the use
of N60 medium for regeneration (Chu et al., 1975). Other
reports on plant regeneration from rice protoplasts included
the work by Onoo et al. (1985), Yamada and Yang (1985),
Coulibaly and Derarly (1986), and Hayashi et al. (1986).
Baba et al. (1986) were the first to transform rice
protoplasts by using spheroplasts to introduce Ti plasmids
into cells. Rapidly growing colonies of protoplast derived
calli were obtained on hormone free MS medium when
Agrobacterium tumefaciens spheroplasts were introduced into
protoplasts by polyethylene glycol treatment. The successful
transfer of foreign DNA into rice protoplasts and stable gene
expression in transgenic callus was demonstrated by Uchimiya
et al. (1986). Transformation frequencies of 2 to 3% were
achieved by using polyethylene glycol and a chimerical gene
consisting of the nopaline synthase promoter, the
aminoglycoside phosphotransferase II structure gene from Tn5,
and the terminator region from cauliflower mosaic virus. 0 u~
Lee et al. (1986) demonstrated gene expression in rice
protoplasts after introduction of a gene by electroporation.
They successfully introduced chloramphenicol acetyl
11
transferase genes with various promoters into rice
protoplasts.
II. FACTORS AFFECTING RICE TISSUE CULTURE
A great deal of information has been published to
indicate that successful rice tissue culture depends on
several factors. These factors include the genotype of the
donor plant, the composition of the culture media,
pretreatment of explants, culture conditions such as
temperature and light, and physiological status of the donor
plant.
II. 1. Culture Media
Rice explants respond to a wide range of callus induction
and plant regeneration media. The major differences in these
media are the concentrations of macronutrients, their
oxidation states (i.e., ammonium vs. nitrate nitrogen),
and the phytohormone content. Miller's medium (1961) was
widely used in the late 1960's and early 1970's (Niizeki and
Onoo, 1968; Nitsch, 1969; Chen et al., 1974; Wang et al.,
1974). Other nutrient media in wide use for rice tissue
culture include MS (Murashige and Skoog, 1962) and modified
MS media (Chen, 1977), LS (Linsmaier and Skoog, 1965) and
modified LS media (Chaleff and Stolarz, 1981), N 6 medium (Chu
et al., 1975), B5 (Gamborg et al., 1968), and modified
White's medium (Chen et al., 1974; Chu, 1978; Genovesi and
Magill, 1979; Chen et al., 1982; Tsay et al., 1982; Zapata et
12
al., 1982; Chu and Zhang, 1985; Chu et al., 1986). At
present, it appears that a range of basic media work well for
rice tissue culture, but there are indications that genotypes
may differ somewhat in their responses to different media.
SK medium (Chen et al., 1978) and Universal medium (Yang et
al., 1980) have been recommended as suitable media for indica
genotypes. Also it has been shown that providing most of the
inorganic nitrogen in the form of ammonium rather than
nitrate is favorable for rice tissue culture. A
concentration of 3.A mM ammonium in the culture medium was
reported as optimal for indica rice anther culture by Huang
et al. (1978).
Sucrose, glucose, or fructose are typically used as
carbon sources and sometimes osmotic agents in culture media.
Various concentrations of sugar have been tested in rice
tissue culture ranging from 1% - 15%. Chen (1978) reported
that sucrose concentrations of 3-9% increased callus
formation and subsequent organogenesis. However, a low
concentration of sucrose (3%) produced better androgenic
initiation and induction of callus than 6 % sucrose (Chen et
al., 1974; Clapham, 1973; Hu et al., 1978a, b). Chaleff and
Stolarz (1981) obtained improved results in anther culture
using sucrose concentrations of 4-5%, and suggested that the
increased osmotic pressure of the culture medium may be
partly responsible. Other researchers recommend sucrose
concentrations of 6 -8 % for dedifferentiation media and 4-5%
for differentiation media to maximize callus induction and
13
plant regeneration (Res. Group of Rice, SAAS, 1976; Zhang,
1982; Chu et al., 1984; Chu and Zhang, 1985; Chu et al.,
1985; Zhang and Chu, 1985; Chu et al., 1986).
Plant growth regulators are important in rice tissue
culture, especially auxin and cytokinins. Various
combinations of auxins and cytokinins in the culture media
have been tested for both callus induction and plant
regeneration in rice (Niizeki and Onoo, 1968; Harn, 1969;
Nishi and Mitsuoka, 1969; Guha et al., 1970; Iyer and Raina,
1972; Woo and Tung, 1972; Mukherjee, 1973; Wang et al., 1974;
Chen, et al., 1974). In the early 1970's, a 2,4-D
concentration of 9 urn was shown suitable for callus induction
(Chen et al., 1974). NAA appears to be superior to 2,4-D in
differentiation media (Niizeki and Onoo, 1971). Combining
NAA with kinetin resulted in significant increases in callus
induction and plant regeneration rates (Hu and Liang, 1979;
Chen et al., 1982). Other auxins such as 2-methyl, 4-
chlorophenoxyacetic acid (MCPA) and 2,4,5-trichlorophenoxy-
acetic acid (2,4,5-T) were also used for callus induction
(Chou et al., 1978; Liang, 1978; Hu et al., 1978a, b).
Cytokinins, particularly kinetin, appear to help trigger
morphogenic differentiation of callus into plants.
Regeneration rates as high as 78% were obtained in anther
culture when kinetin and NAA were used in the medium (Chen,
1977; Chaleff and Stolarz, 1981; Lee and Chen, 1982; Zhang,
1982). Wakasa (1982) substituted 6 -benzylaminopurine (BA)
for kinetin in differentiation media, and also reported a
14
high rate (67%) of green plantlet regeneration.
II. 2. Genotype
The genotype of donor plants in rice tissue culture
appears to be the most important factor in callus induction
and plant regeneration. Genotypic differences in anther
culture have been reported for rice. Niizeki and Oono (1968)
cultured anthers of 10 japonica varieties. Only six
varieties formed callus, and only the varieties Nonling 20
and Toride 2 produced green plantlets, indicating genotypic
differences for tissue culture traits among japonica
varieties. Mukherjee (1973) noticed genotypic differences in
the in vitro formation of embryoids from rice pollen.
Differences existed not only between ecogeographic races of
rice, but also among cultivars within the same species.
Similar findings were also reported by various other research
groups (Chen et al., 1974; 1974; Oono, 1975; Chen and Lin,
1976; Yin et al., 1976; Chen, 1978; Woo et al., 1978;
Cornejo-Martin and Primo-Millo, 1981).
Culturability is defined as the product of the callus
formation rate times the plant regeneration rate, and is
typically expressed as a percentage. Sheng et al. (1982)
suggested that culturability differed between major types of
rice in the following order: glutinous rice > japonica >
indica/japonica hybrids > japonica/indica hybrids > hybrid
rice > indica. The culturability of Oryza sativa was higher
than that of 0 . perennis (annual type), and wild rice only
15
produced albino plants (Wakasa, 1982). Similar findings were
obtained by Woo and Huang (1980), where interspecific crosses
of 0 . sativa with 0 . perennis and 0 . sativa with 0 .
glaberrima showed reciprocal differences in anther
culturability. Ding et al. (1983) evaluated the anther
culturability of 38 indica varieties. Different responses in
callus formation rate ranging from a low of 0.35% to a high
of 28.4% were reported. Plant regeneration rates among 38
genotypes followed a similar pattern.
Abe and Sasahara (1982) suggested that culturability was
dependent on genotype. They provided evidence for the
genetic control of culturability using cultivars of high and
low callus formation rates and the F^ from their
hybridization. Calli induced from seeds showed a clear
resemblance to the parent of low culturability. They
suggested that factors suppressive to callus formation were
genetically dominant.
Xui and Liu (1984) analyzed the genetic effects of
culturability by diallel analysis. Significant differences
in terms of general combining ability (gca) and specific
combining ability (sea) were found, indicating a strong
effect of the donor genotypes. Zhang and Chu (.1985) and Chu
(1986) made a complete 5 x 5 diallel cross to study the
genetic background of callus formation and plant
regeneration. Their results indicated that maternal effects,
additive effects, and slightly high overdominance contribute
to genotypic differences. Miah et al. (1985) studied the
16
inheritance of callus formation rate for 4 parents and 1 0 Fj
hybrids. Diallel analysis indicated that strong genotypic
differences existed among the parental array. Culturability
was inherited as a recessive character controlled by a single
block of genes. Similar findings were reported by Boyajiev
and Kuong (1986).
Chu et al. (1986) cultured stem node callus derived from
1 2 wild species of Oryza and 2 interspecific hybrids.
Regeneration was obtained in 9 species and culturability
ranged as high as 17.76%. The study indicated genotypic
differences among rice species. Similar results were found
using tetraploid plants (Chu et al., 1985).
Genotypic differences in callus formation and plant
regeneration were recently reported by Davoyan (1987).
Tissue culture of 0. rufipogon, tetraploid 0. latifolia, and
32 lines, 57 varieties, and 14 mutants of 0. sativa indicated
that callus formation and morphogenesis depended on genotype.
In this case, it was suggested that the genetic factors
controlling callus formation and regeneration capacity were
independent.
Analysis of proteins from embryogenic and non-
embryogenic calli of rice indicated that two polypeptides of
54 and 24 kd present in embryogenic callus corresponded to
the major polypeptides in embryo extracts. This result
suggested that certain polypeptides may be good indicators of
regeneration capacity in rice (Chen and Luthe, 1987).
17
II.3. Culture Conditions and Pre-treatments
Two aspects of the culture environment which are
relatively easy to control are temperature and light. The
temperature used for callus induction in rice anther culture
is generally 25° C. Wang et al. (1978) suggested that the
temperature used for callus induction influenced the
regeneration frequency for green and albino plants. With a
slightly higher temperature, the number of albino regenerated
plants increased. In general, temperatures of 25° C or
slightly less are widely used (Chen et al., 1982; Zhang,
1982; Chen et al, 1982).
The light conditions used for rice anther culture range
from complete darkness to continuous illumination. For
callus induction, inoculated anthers were typically placed in
the dark (Niizeki and Oono, 1968; Harn, 1969; Woo and Tung,
1972; Mukherjee, 1973; Chen et al., 1974). However, Nishi
and Mitsuoka (1969) and Niizeki and Oono (1971) also reported
callus induction under continuous light. The generally
accepted current practice is to use dark incubation for
callus induction followed by 16 hours light per day at 2 0 0 0 -
3000 lux for plant regeneration (Chen et al., 1982; Zhang,
1982; Zapata et al, 1982; Croughan et al, 1983, 1984, 1985,
1986; Zhang and Chu, 1984; Chu and Zhang, 1985; and Zhang and
Chu, 1985).
Physical and chemical pretreatment of anthers and
panicles has proved effective in enhancing callus formation
and regeneration frequencies. Cold temperature treatment of
18
anthers before inoculation substantially increases callus
formation (Wang et al., 1974; Chaleff et al., 1975). Cold
pretreatments have become a broadly adopted procedure for
rice anther culture. Anthers are incubated for 1 to 12 days
at 2 to 13° C before inoculation onto callus induction medium
(Hu, 1978; Genovesi and Magill, 1979; Chaleff and Stolarz,
1981; Cornejo-Martin and Primo-Millo, 1981; Zhang, 1982;
Zapata et al., 1982; Qu and Chen, 1983a, b; Croughan et al.,
1983, 1984, 1985). The combination of the temperature and
duration of the pretreatment varied among these experiments.
Other pretreatments which have been tested include
irradiation, centrifugation, and exposure to magnetic fields.
Centrifugation of rice panicles at 2,000 rpm for 10 minutes
reportedly enhanced the rate of callus formation and plant
regeneration (Zhu and Wang, 1982). Sun et al. (1978)
reported that callus formation rate was increased following
gamma ray irradiation with a dosage of 100 R. Recent
experiments indicated that indica rice exposed to magnetic
fields may produce callus at higher rates (GuangXi Acad.
Agric. Sci., personal communication).
Chemical treatment with Etherel (2-chloroethylphosphoric
acid) has proved effective for inducing callus formation.
Spraying the donor plant with 0.2-0.4% Etherel at the meiotic
stage of pollen development enhanced the subsequent response
to callus induction medium (Wang et al., 1974; Lab of
Physiol. Acad. Sin., 1975). The combination of cold
temperature and Etherel treatment further increased the rate
19
of callus formation (Hu et al., 1978). However, chemical
treatment before explant inoculation is not generally
utilized in rice tissue culture.
III. SOMACLONAL VARIATION IN RICE
Genetic variation derived from both diploid and haploid
culture of rice has been observed for a wide range of
agronomic traits. The ability to enhance the level of
genetic variability and even the possibility of producing
novel genes is exciting to plant biologists in general and
provides new experimental options for plant improvement
(Larkin and Scowcroft, 1981; Scowcroft and Larkin, 1982).
Several papers have been published to indicate the importance
of utilizing the induced variability for varietal improvement
in rice.
III. 1. Somaclonal Variation in Diploid Culture
Oono (1978) was the first to present an extensive study
on rice somaclonal variation. The progeny of selfed
regenerants derived from the culture of rice seeds showed a
wide range of variation in plant height, flowering date,
plant type, spikelet fertility, and chlorophyll content.
Among 1121 somaclones derived from 75 calli, only 28.1% of
the plants expressed the normal parental phenotype for the
above mentioned 5 characters. Twenty-eight percent of the
plants were variant for more than 2 characters. He further
estimated that mutations governing these 5 characters were
20
induced in the culture process at the rate of 0.03 - 0.07
mutations per cell per division. Similar findings for
somaclonal variability were reported by Fukui (1980) and Zhao
et al. (1982).
Sun et al. (1983) conducted a genetic study on
somaclonal variation in rice. Analysis of 950 T2 (R2) lines
revealed significant genetic variability for the 5 characters
of plant height, effective tillering, grain number per
panicle, heading date, and 1,000 grain weight. Only 24.2% of
the somaclones expressed the normal parental phenotypes, a
finding similar to that of Oono (1978). These reports
provided strong evidence that during the process of callus
formation and plant regeneration mutations were induced in
rice. It is unlikely that pre-existing variability due to
incomplete homozygosity was a factor since the donor plant
underwent 7 cycles of selfing with protective bagging
beforehand.
Zhang and Chu (1984) utilized a modified system of rice
somaculture which included the step of immature panicle
culture of haploid plants (n=1 2 ) derived from anther culture.
In this case, heterozygosity and heterogeneity should be
eliminated following artificial doubling of the chromosomes.
Phenotypic and chromosomal variability existed in both the
SS2 (R2) and SS3 (R3) generations. Variation in plant
height, panicle length, grain number per panicle, and panicle
number per plant was observed between and within somaclonal
lines.
21
III. 2. Somaclonal Variation in Haploid Culture
Morphological variations among regenerated plants from
rice haploid culture were first reported by Woo et al.
(1973). They described variations in culm length, spikelet
fertility, grain size, number of tillers, and growth habit in
the T2 generations of dihaploid plants derived from anther
culture of hybrids. Russo et al. (1983) found that there
were significant differences in yield and protein content in
addition to morphological variations among 16 diploid lines
derived from anther culture of plants. Whether the
variations noticed in these two cases were pollenclonal in
nature was not clearly elucidated, because the donor plants
were genotypically heterozygous.
Chu et al. (1984) used a homozygous genotype derived
from anther culture and reported pollenclonal variation in
the second culture cycle. Forty-four pollen lines showed
significant differences from the parent SH3 in 6 agronomic
characters including plant height, panicle length, flag leaf
length, grain number per panicle, heading date, and 1 , 0 0 0
grain weight. The coefficient of variation (0.35-0.83) was
significantly higher than that for the parent donor (0.07-
0.14). Among the pollenclones, 26 were diploids and 15
haploids, with 1 trisomic and 1 tetrasomic; while all
parental plants were diploids. Meiotic chromosomal
abnormality exceeded 16% for pollen clones while the value of
the SH3 parent was 5%.
Cytogenetic studies on pollenclonal variation in rice
22
have been reported (Chu et al., 1984; Chu, 1985; Chu and Chu,
1985; Chu and Zhang, 1985; Chu et al., 1985). In a study of
290 pollenclones derived by anther culture of 34 hybrids
and 19 parents, morphological variation was correlated to
cytogenetic abnormalities. The characters of plant height,
growth habit, and leaf morphology in pollen plants varied
significantly and corresponded to chromosomal abnormalities
such as loose pairing, laggards, straggling chromosomes,
bridges, and fragments in pollen mother cells. Aneuploids,
including primary trisomics, tertiary trisomics, tetrasomics,
monosomies, and nullisomics, were also found in pollenclones
derived from anther culture (Chu et al., 1985).
Morphological variation has been found in the progeny
derived from anther culture of tetraploid rice. Chu et al.
(1986) described variability in plant height, awns, grain
size, and tiller number for somaclones derived from
tetraploid anthers.
III. 3. Somaclonal Variation in Biochemical Traits
Biochemical variations have been produced through tissue
culture using mutagenic treatments and cellular level
selection. Chen and Chen (1979, 1980) selected 5-
methyltryptophan (5MT) resistant pollen calli from which
resistant plants were regenerated. Selfed progenies of the
resistant plants segregated for the resistance trait
producing highly resistant, moderately resistant, and
sensitive plants. This trait appeared to be controlled by a
23
semidominant gene (Chen, 1981). Schaeffer and Sharpe (1981)
reported the selection of calli resistant to aminoethyl-L-
cysteine (AEC) in culture medium. Plants regenerated from
the selected calli produced seeds containing higher levels of
lysine, alanine, arginine, and asparagine than the controls.
Selections for salt tolerance in rice tissue culture
have been reported by many researchers. Oono (1977) selected
diploid rice lines resistant to sodium chloride. Tolerance
was maintained in some regenerants and appeared to be
inherited through 3 generations. However, the segregation
pattern of this trait was complicated. Several studies have
reported selection of salt and sea water tolerant callus
(Croughan et al., 1981; Anonymous, IRRI, 1981; Heyser and
Nabors, 1982; Yano et al., 1982; Yasuda et al., 1982; Wong et
al., 1983; Ye, 1984; Woo et al., 1986; Zapata et al., 1986;
Kishor and Reddy, 1986).
Selection for disease resistance through exposure of
anther derived tissue to phytotoxins has been reported (Chu,
1984; Zheng and Chu, 1985). Successful plant regeneration
was obtained from callus cultured on medium containing
culture filtrate of FI and E3 races of rice blast
(Pyricularia oryzae) (Zheng et al., 1985). Field tests
entailing exposure to various physiological races of blast
indicated that advanced progeny of somaclones possessed
resistance (Zheng and Chu, 1985; Zheng et al., 1985).
Phytotoxins have also been used for in vitro selections
for resistance to brown spot disease (Helminthosporiuro
24
oryzae) by Ling and colleagues (1985, 1986). In a study of 5
varieties, callus derived from immature panicles was screened
using media containing 10%, 25%, and 50% by volume culture
filtrate. Very small pieces of callus were shaken in toxin
supplemented medium for 2 days before subculture to toxin-
free medium. Two plants which regenerated from the 25% toxin
treatment proved resistant to the pathogen.
Sun et al. (1986) also reported in vitro selection of
Xanthomonas oryzae resistant mutants in rice. Of 365 calli
derived from seeds of the susceptible variety Nangeng 34 and
inoculated with X. orzyae strain Ks8-4, 63 calli showed
sectional proliferation, producing 45 regenerants. All but
one were resistant and produced resistant progenies through 3
generations.
Other biochemical and physiological variations reported
for rice tissue culture included amino acid analog resistance
(Chaleff and Stolarz, 1981), variation in protein content
(Shaeffer et al., 1984), protein polymorphism (Russo and
Raso, 1983), chilling resistance (Li, 1980), and nitrate
reductase deficiency (Wakasa et al., 1984).
IV. VARIETAL IMPROVEMENT THROUGH RICE TISSUE CULTURE
IV. 1. Use of Anther Culture in Rice Breeding
Since 1970, excellent progress has been made in applying
anther culture techniques to rice varietal improvement. In
1975, China established a national anther culture breeding
program involving 20 institutions coordinated by the Shanghai
25
Academy of Agricultural Sciences. The first varieties from
anther culture were released in 1976 and included Xin Xiou
(Shanghai Academy of Agricultural Sciences), and Huayu
(Tienjin Inst, of Rice Res. and the Inst, of Genet., Acad.
Sin.). According to information from the National Symposium
on Rice Anther Culture in Nanchang in 1982, 25 varieties from
anther culture had been released by 1982 and were planted on
30,000 ha. This figure rose to 28 varieties by 1983 (Sheng
et al., 1983). Zhang and Chu (1986) summarized anther
culture breeding in China and reported over 100 varieties and
breeding lines developed through anther culture. The authors
proposed a standard sequence for anther culture entailing 1 )
parental selection, 2) hybridization of the parents, 3)
anther culture, A) genetic evaluation and selection of
superior lines in H2 and H3 generations, and 5) yield testing
at multiple locations.
Recently, anther culture techniques have been used to
breed for disease resistance (Zheng and Chu, 1985; Ling et
al., 1985, 1986; Sun et al., 1986), cold tolerance (Kato et
al., 1986), aluminium-tolerance (Okawara et al., 1986),
improved grain quality (Shaeffer et al., 198A), and salinity
tolerance (Zapata et al., 1986, Chen, 1986a, b).
IV. 2. Use of Somaclonal Variability in Rice Breeding
Somaclonal variability has been found among the
regenerates from a long list of cultivated rice varieties
(Oono, 1978; Zhao et al., 1982; Sun et al., 1983; Zhang and
26
Chu, 1984). The rice variety T42 was developed in China
through somaculture and released in 1983 (Zhang and Chu,
1986).
High-yielding clones have been established via tissue
culture of immature panicles of elite tetraploids derived by
hybridization. In yield trials in 1982 and 1983, two lines
were superior to their diploid control. Apart from total
yield increases, considerable improvements in 1 , 0 0 0 grain
weight have been achieved and plant height has been reduced
by up to 40 cm (Chen et al., 1987).
CHAPTER I
GENETICS OF PLANT REGENERATION IN IMMATURE PANICLE
CULTURE OF RICE (ORYZA SATIVA L.)
ABSTRACT
A total of 6,332 rice immature panicles derived from 10
cultivars, 12 F^ hybrids from a 4 x A diallel cross, their F2
and F 3 progenies, and 24 BCF^ hybrids were cultured iri vitro.
Genotypic differences in plant regeneration and inheritance
of this trait were investigated based on generation mean
analysis.
Significant genotypic differences in regeneration rate
were found among the 10 varieties. The variety 'Lemont'
possesses a high regeneration rate, averaging 6 . 6 plants per
panicle, and this character was inherited by its progenies
(BCF^, F 2 > In contrast, the indica rice variety 'IR36'
and its progeny have low regeneration rates. Generation mean
analysis of regeneration reveals significant values for
dominance gene effects (d) and epistatic effects (additive x
additive, additive x dominance, dominance x dominance).
Additive gene effects (a) appeared important in certain
crosses. The heritability estimate (h) calculated by parent-
off spring regression is 0.63, indicating that this trait is
heritable.
27
28
Additional index words: tissue culture, diallel, parent-
off spring regression, heritability, generation mean analysis.
INTRODUCTION
Rice immature panicles have been widely used as an
explant source to study embryogenesis, plant regeneration,
and somaclonal variation (Tang, 1979; Su, 1980; Zhao et al.,
1982; Ling et al., 1983a, b; Sun et al., 1983; Zhang and Chu,
1984; Chen et al., 1985; Croughan et al., 1986). Higher
regeneration rates have been reported for in vitro culture of
immature panicles compared to other explant sources.
Somaclones derived from immature panicle culture possess a
wide range of genetic variability, including changes in
maturity, plant height, plant type, and panicle size (Sun et
al., 1983; Zhang and Chu, 1984). Such somaclones offer a
potentially useful source of genetic variability for use in
varietal improvement (Zhang and Chu, 1986; Croughan et al.,
1986).
The capacity to regenerate sufficient numbers of
somaclones to produce adequate population sizes from which to
select is an important component of utilizing somaclonal
variation for varietal improvement. Efforts at increasing
29
plant regeneration rates have primarily concentrated on
screening various culture media (Ling et al., 1983a) and
sampling various explant materials (Zhang and Chu, 1984; Chen
et al., 1985). The genetic aspects of plant regeneration are
generally considered important, but have been less widely
studied. Genotypic effects on plant regeneration in rice
have been reported for anther culture (Chu et al., 1984; Chu
et al., 1985; Zhang and Chu, 1986), stem node culture (Chu et
al., 1986), and protoplast culture (Yamada et al., 1986). No
studies on the genetics of plant regeneration in immature
panicle culture have been reported.
The present study, based on immature panicle culture of
nine indica rice varieties and the U.S. rice cultivar Lemont,
attempted to clarify genotypic differences in regeneration
among a range of rice cultivars; to elucidate the genetic
background of the trait based on a 4 x 4 diallel cross and
evaluation of the F^, F 2 , F 3 , and BCF3 generations; to
analyze various gene effects which contribute to the variance
of the generation means, and to estimate the heritability of
the trait by parent-offspring regression analysis.
MATERIALS AND METHODS
Sampling
The varieties were selected to represent a broad genetic
collection from diverse geographic origins, as presented in
30
Table 1. Three weeks after sowing, seedlings at the A-leaf
stage were transferred from the greenhouse to the field and
hand transplanted in a complete randomized block design with
six blocks consisting of 15 replicates per block. Shoots
were randomly collected in the boot stage when the tip of
flag leaf emerged 3 to 5 cm above the penultimate leaf
collar. Leaf blades were removed before sealing the culms in
plastic bags for incubation at 4° C for 3 days.
Immature panicle culture
Culms were surface sterilized by soaking for 30 minutes
in 2.5% sodium hypochlorite (50% solution of commercial
bleach in distilled water), and rinsed twice with sterile
distilled water. Following aseptic excision from the culm,
young panicles 0.5 to 3 cm in length were transferred to
individual 100 x 60 mm disposable petri dishes containing R4
medium (Chaleff and Stolarz, 1981). Dishes were sealed with
Parafilm and incubated in the dark at 25° C for 20 days, then
transferred to a 16:8 hour fluorescent light photoperiod.
Hybridization
Based on the regeneration rates of the 10 varieties
tested, the four varieties Lemont, IR36, Guichow1 and Short
Tetep' were identified as representing a range of
regeneration rates and used as parents for a A x 4 diallel
cross. Seed of each parent was planted in the greenhouse at
7-day intervals for 6 weeks to provide synchronized flowering
31
for hybridization. Crosses were made by hand pollination
following vacuum emasculation and glysine bagging of the
panicle the previous afternoon. Progeny plants were screened
for expression of appropriate parental traits, the selfed
progeny eliminated, and the Fj hybrids identified for
subsequent backcrossing to parents (Appendix 1). The F2 and
F3 populations from each cross were obtained by selfing. A
total of seven populations including parents (P3 and P2 )>
BCF^ (to both parents), F2 , and F3 , were established to study
the genetics of plant regeneration in immature panicle
culture.
Statistical analysis
Regeneration rate in immature panicle culture was
calculated as number of regenerants/number of panicles plated
x 100%. Genotypic differences among the ten varieties were
calculated by comparison of the means. The Duncan's new
multiple-range test of different means were compared using
the equation by Steel and Torrie (1980).
Based on generation mean analysis (Gamble, 1962), various
gene effects were calculated in terms of mean gene effect
(m), additive effect (a), dominance (d), additive x additive
(aa), additive x dominance (ad), and dominance x dominance
(dd). Six equations for calculating various gene effects are
presented in Appendix 2, Table 2. The significance of these
gene effects were calculated on the basis of the variance of
the population means. The equations for testing the
32
significance of various estimates are presented in Appendix
2, Table 3. The significance of each estimate was calculated
by t-test of the standard deviation to the estimates.
RESULTS AND DISCUSSION
I. Genotypic differences in plant regeneration rates from
immature panicle culture.
A wide range of plant regeneration rates was found in
immature panicle culture of the 10 varieties tested (Table
1). The varieties IRGA 409' and Lemont produced the highest
average rates (7.06 and 6.60 plants per panicle,
respectively) while Cica 8 ' produced the lowest rate. The
varieties Short Tetep, Tetep, and Gui Chow produced
intermediate rates of plant regeneration. Analysis of
variance and comparison of means for plant regeneration rates
indicated significant differences between genotypes (Table
1) .
The genotypic differences in regeneration rates using
immature panicles as the explant source are in agreement with
results from previous studies where various other explants
were used (Zhang and Chu, 1985; Miah et al., 1985; Chu et
al., 1986). The regeneration rates of Lemont and IRGA 409
are higher than some japonica varieties, although Lemont is a
long grain U.S. variety and IRGA 409 is an indica type. The
order of culturability proposed by Sheng et al. (1982) of
33
glutinous > japonica > indica/japonica > hybrid rice >
indica, therefore, may have exceptions.
II. Plant regeneration rates in a 4 x A diallel cross and the
BCF^, F2 , and F 3 progeny.
The varieties Lemont, IR36, Short Tetep and Gui Chow
were selected as parents for a complete 4 x 4 diallel cross.
Lemont represented a high rate of plant regeneration, Short
Tetep and Gui Chow intermediate rates, and IR36 a low
regeneration rate. All Fj hybrids expressed regeneration
rates similar to their high parents (Table 2), and no
significant differences were found in the corresponding
reciprocals (t-test), indicating that maternal effects were
not important, at least for these varieties.
Table 3 presents the mean and range of regeneration
rates in the 12 F 2 and F3 populations. The mean values of F2
populations are lower than F 3 values except IR36/Short Tetep,
IR36/Gui Chow, Gui Chow/IR36, and Gui Chow/Short Tetep, which
show no significant difference between Fj and F2 means (t
test). This may be due to the crosses having one low parent.
A similar pattern for both mean and range in a decreasing
order was found in F 3 populations.
Regeneration performance was evaluated in 24 backcrosses
(Table 4). The mean value of BCF^ increases when backcrossed
to the high parent, and decreases when backcrossed to the low
parent. An example of this pattern can be found in comparing
the cross Lemont/lR36//Lemont (m = 4.80) with
34
Lemont/lR36//IR36 (m = 3.51).
The results of this study indicate that regeneration
rates among the 1 2 hybrids studied tended to resemble the
rate of their higher parent. This is in contrast to the
findings by Abe and Sasahara (1982) where calli derived from
F^ seeds showed a clear resemblance to the parent with the
lower plant regeneration rate. The results are also opposite
to the findings by Miah et al. (1985) and Zhang and Chu
(1985), where the regeneration rate in anther culture of F^
hybrids appeared to be recessive. It therefore appears that
explant-specific gene regulation may be involved.
III. Estimates of gene effects.
The mean values for the plant regeneration rates of the
various populations is shown in Table 5. In describing the
cross, the female parent is designated as Pj, and the male
parent as P2 . Pl^i is the backcross of the F^ to the female
parent, and P2 ^i is the backcross to the male parent.
Estimates of the six parameters for gene effects by
population means is presented in Table 6 . The significant
estimates have been indicated by t-test of their standard
deviation to the estimates at the 0.05 and 0.01 probability
levels.
The major contribution by dominance gene effects to
variation among the 1 2 crosses is indicated by the relative
magnitude of dominance to mean. The estimate of dominance
effects is highly significant in all crosses except
35
Lemont/Short Tetep, Short Tetep/Lemont, and Gui Chow/Lemont.
Seven of the 12 crosses show significant values for
additive gene effects, with the values negative except in the
case of Lemont/Short Tetep. The relative magnitude of
additive gene effects is generally less than that of the
dominance gene effects, suggesting that the contribution of
additive gene effects in general is not as important as that
of dominance effects.
Epistatic gene effects, aa, ad, and dd estimates,
although of minor importance in certain crosses, are
significant in the inheritance of plant regeneration. Seven
of 1 2 crosses showed significant values for additive x
additive gene effects with the value negative except for
Lemont/IR36. Five of 12 crosses had significant values for
additive x dominance effects. Nine of 12 crosses had highly
significant values for dominance x dominance gene effects,
with positive values except for ST/Lemont and ST/IR36,
suggesting an increasing effect on inheritance. Significance
of aa, ad, and dd estimates strongly indicate that epistatic
gene effects play an important role in the inheritance of
regeneration.
Generation mean analysis of the various gene effects on
regeneration provides genetic information on the relative
magnitudes of the contributions of additive, dominance and
epistatic effects to inheritance of the trait. Significant
epistatic and dominance gene effects contribute more to the
total variance, although additive effects are of minor
36
importance for some crosses.
IV. Heritability estimates.
Based on parent-offspring regression analysis (Smith and
Kinman, 1965), heritability is estimated as b/(2rxy), where
rxy is a measure of the relationship between the parent y and
its offspring x, and b is the regression of y on x.
Calculation of the middle parent value to its F^ indicates a
high regression coefficient, and the heritability estimate
for plant regeneration rate is 0.63. Calculation of F 2 to
the corresponding F 3 values indicated a regression
coefficient of 0.38 and a heritability estimate for plant
regeneration rate of 0.25. Hybridization is therefore an
efficient method for transferring high regeneration rates
into germplasm lacking this character.
37
REFERENCES
Abe, T., and T. Sasahara. 1982. Genetic control of callus
formation in rice. p. 410-420. In A. Fujiwara (ed.) Plant
tissue culture. Muzen, Tokyo.
Chaleff, R.S., and A. Stolarz. 1981. Factors influencing the
frequency of callus formation among cultured rice (Oryza
sativa) anthers. Physiol. Plant 51:201-206.
Chen, T., L. Lan, and S. Chen. 1985. Somatic embryogenesis
and plant regeneration from cultured young
inflorescences of Oryza sativa L. (rice). Plant Cell
Tissue Organ Cult. 4:51-54.
Chu, Q.R., P.J. Xi, and Z.H. Zhang. 1984. Pollenclonal
variation of rice (Oryza sativa L.). Shanghai Agric. Sci
Tech. 4:22-24.
Chu, Q.R., Y.H. Gao, and Z.H. Zhang. 1985. Cytogenetical
analysis on aneuploids from pollenclones of rice (Oryza
sativa L.). Theor. Appl. Genet. 71:506-512.
Chu, Q.R., H.X. Cao, Y.Q. Gu, and Z.H. Zhang. 1986. Stem node
culture of 1 2 wild species and two distant hybrids in
Oryzae. Acta Agric. Shanghai 2 (l):39-46.
Croughan, T.P., Q.R. Chu, and M.M. Pizzolatto. 1986. The use
of anther culture to expedite the breeding and release of
new varieties of rice. 78th Ann. Res. Rep., Rice Res.
Stn. p. 38.
38
Gamble, E.E. 1962. Gene effects in corn (Zea mays L.). II.
Relative importance of gene effects for plant height and
certain component attributes of yield. Can. J. Plant Sci.
42:349-358.
Ling, D.H., W.Y. Chen, M.F. Chen, and Z.R. Ma. 1983a. Direct
development of plantlets from immature panicles of rice
in vitro. Plant Cell Report 2:172-174.
Ling, D.H., W.Y. Chen, M.F. Chen, and Z. R. Ma. 1983b.
Somatic embryogenesis and plant regeneration in an
interspecific hybrid of rice. Plant Cell Report 2:169-
171.
Miah, M.A.A., E.D. Earle, and G.S. Khush. 1985. Inheritance
of callus formation ability in anther culture of rice,
Oryza sativa L. Theor. Appl. Genet. 70:113-116.
Sheng, J.H., M.F. Li, Y.Q. Chen, and Z.H. Zhang. 1983.
Improving rice by anther culture, p.183-205. In Hu, H.
(ed.) Cell and tissue culture in cereal crop improvement.
Science Press, Beijing.
Su, L.H. 1980. Plant regeneration from immature panicle
culture of rice. J. Wuhan Univ. 3:37-46.
Smith, J.D., and M.L. Kinman. 1965. The use of parent-
off spring regression as an estimator of heritability.
Crop Sci. 5:595-596.
Steel, R.G.D., and J.H. Torrie. 1980. Principles and
procedures of statistics, p. 107-115. 2nd edition,
MxGraw-Hill, Inc. New York.
Sun, Z.X., C.Z. Zhao, K.L. Zheng, S.F. Qi, and Y.P. Fu.
39
1983. Somatic genetics of rice (Oryza sativa L.). Theor.
Appl. Genet. 67:67-73.
Tang, Y.Y. 1979. Regeneration from immature panicle culture
of rice. Acta Phytophysiol. Sin. 4:35-42.
Yamada, Y . , Z.Q. Yang, and D.T. Tang. 1986. Plant
regeneration from protoplast-derived callus of rice
(Oryza sativa L.). Plant Cell Reports 5:85-88.
Zhang, L.N., and Q.R. Chu. 1984. Characteristical and
chromosomal variation of rice somaclones. Sci. Agric.
Sin. 18 (4):32-40.
Zhang, Z.H., and Q.R. Chu. 1985. Biometrical analysis on
anther culturability in rice (Oryza sativa L.). Acta
Agric. Shanghai 1 (3):1-10.
Zhang, Z.H., and Q.R. Chu. 1986. Advances in rice anther
culture for varietal improvement in China. J. Agric.
China 2(suppl.):10-16.
Zhao, C.Z., K.L. Zheng, X.F. Qi, Z.X. Sun, and Y.P. Fu. 1982.
Characteristics of rice plants derived from somatic
tissue and their progenies in paddy field. Acta Genet.
Sin. 9:320-324.
40
Table 1. Genotypic differences in plant regeneration from immature panicle culture of 1 0 rice varieties.
Variety and F]_'s Origin
Number of immature panicles plated
Number of regenerated plants per plate (mean) s.d.
IRGA 409 Brazil 16 7.06 a 2.04
Lemont U.S.A. 371 6.60 a 4.63
Short Tetep U.S.A. 117 2.52 b 3.00
Tetep Vietnam 53 1.28 be 0.98
Gui Chow China 336 1.14 be 0.80
Costa Rica 1113 U.S.A. 2 1 2 0.63 c 0.80
Nanj ing 11 China 223 0.37 c 0.61
Cica 6 Colombia 187 0 . 2 0 c 0.43
IR36 Philippines 1 2 1 0.14 c 0.28
Cica 8 Colombia 1 2 0 . 0 1 d 0 . 1 0
* Means with the same letter are not significantly different at the 0.05 level according to Duncan's multiple range test.
41
Table 2. Plant regeneration rates in 4 x 4 diallel cross.
Parents and Number of immature Average number oftheir F^'s panicles plated regenerated plants
per panicle
Lemont 371 6.60Lemont/lR36 81 7.28Lemont/Short Tetep 72 6.70Lemont/Gui Chow 72 6.35
IR36 1 2 1 0.14IR36/Lemont 54 5.65IR36/Short Tetep 33 2 . 6 6IR36/Gui Chow 41 1.31
Short Tetep 117 2.52Short Tetep/Lemont 60 5.68Short Tetep/IR36 34 2.61Short Tetep/Gui Chow 40 3.04
Gui Chow 336 1.14Gui Chow/Lemont 35 4.24Gui Chow/IR36 14 1.46Gui Chow/Short Tetep 28 2.35
Table 3. Plant regeneration from immature panicle culture of F2 and F 3 populations.
Cross
F2 ,Lemont/IR36Lemont/Short TetepLemont/Gui ChowIR36/LemontIR36/Short TetepIR36/Gui ChowShort Tetep/LemontShort Tetep/IR36Short Tetep/Gui ChowGui Chow/LemontGui Chow/lR36Gui Chow/Short Tetep
F3 #Lemont/IR36Lemont/Short TetepLemont/Gui ChowIR36/LemontIR36/Short TetepIR36/Gui ChowShort Tetep/LemontShort Tetep/IR36Short Tetep/Gui ChowGui Chow/LemontGui Chow/IR36Gui Chow/Short Tetep
Regeneration rate
Mean Range
3.12 0-17.64.90 0-17.84.94 0.2-12.35.41 0.2-16.83.46 0-14.11.54 0- 8.25.13 0-10.52.31 0- 8.23.87 0-16.23.65 0-10.83.69 0-15.23.25 0- 9.3
2.17 0- 8.04.68 0-10.04.70 0.3- 9.83.31 0-11.71.81 0 -1 1 . 01.39 0- 3.35.40 0.5-13.61.85 0.3- 3.43.94 0-12.01.40 0.2- 3.32.13 0.1- 4.56.44 0.4-20.3
Number ofimmaturepaniclesplated
29223428026978
108262121195198120109
845953833133874789607725
43
Table 4. Plant regeneration from immature panicle culture of 24 backcrosses.
Cross
Number of immature panicles plated
Regeneration rate
Mean Range
Lemont/IR3 6 //Lemont Lemont/IR36//IR36 Lemont/ST//Lemont Lemont/ST//ST Lemont/Gui Chow//Lemont Lemont/Gui Chow//GuiChow
IR36/Lemont//IR36 IR3 6/Lemont//IR36 IR36/ST//IR36 IR36/ST//ST IR36/GuiChow//lR36 IR36/Gui Chow//GuiChow
ST/Lemont//ST ST/Lemont//Lemont ST/IR36//ST ST/IR36//IR36 ST/Gui Chow//ST ST/Gui Chow//Gui Chow
GuiChow/Lemont//GC GuiChow/Lemont//Lemont Gui Chow/lR36//GC Gui Chow/IR36//lR36 Gui Chow/ST//Gui Chow Gui Chow/ST//ST
92 4.80 0-13.769 3.51 0.1-11.598 5.73 0.1-10.417 3.47 3.1-6.071 4.43 1.6-9.927 5.60 5.60
1 2 2.43 2.4337 5.00 1.0-9.91 0 2 . 1 0 2 . 1 057 2.54 0 -1 1 . 864 0.39 0-2.554 1.81 0-9.4
98 3.29 0-10.448 7.42 0.7-21.365 2.85 0 -1 0 . 648 2.06 0-4.571 3.20 0 .6 -8 . 659 1.53 0 -8 . 8
33 1 . 1 2 0.9-4.02 0 5.90 4.6-9.21 0 0 014 0 . 1 0 0 . 114 0.14 0-0.373 1 . 8 6 0-3.2
44
Table 5. Generation means for plant regeneration from immature panicle culture of rice.
Cross PA* ? 2 * 1 * 2 *3 PlFi P2 Fi
Lemont/IR36 6 . 6 0 . 1 7.3 3.1 2 . 2 4.8 3.5
Lemont/ST 6 . 6 2.5 6.7 4.9 4.9 5.7 3.5
Lemont/Gui Chow 6 .6 1 . 1 6.4 4.9 4.7 4.4 5.7
IR36/Lemont 0 . 1 6 . 6 5.7 5.4 3.3 2.4 5.0
IR36/ST 0 . 1 2.5 2.7 3.5 1 . 8 2 . 1 2.5
IR36/Gui Chow 0 . 1 1 . 1 1.3 1.5 1.4 0.4 1 . 8
ST/Lemont 2.5 6 . 6 5.7 5.1 5.4 3.3 7.4
ST/IR36 2.5 0 . 1 2 . 6 2.3 1.9 2.9 2 . 1
ST/Gui Chow 2.5 1 . 1 3.0 3.9 3.9 3.2 1.5
GuiChow/Lemont 1 . 1 6 . 6 4.2 3.7 1.4 1 . 1 5.9
Gui Chow/lR36 1 . 1 0 . 1 1.5 3.7 2 . 1 0 . 0 0 . 1
Gui Chow/ST 1 . 1 2.5 2.4 3.3 6.4 0 . 1 1.9
* Pi = female parent in cross P2 = male parent in cross ?! = P!/P2F2 = selfed seeds from F^F3 = selfed seeds from F2PfFi = Fi backcrossed to female parentP2 F 1 = F^ backcrossed to male parent
45
Table 6 . Mean estimates of six gene effects in plant regeneration rates.
Cross Gene effects
m a d aa ad dd
LMNT/IR36 3.12* 1.29 8.04** 4.14* -1.92* 0.58
LMNT/ST 4.90* 2.26* 0.96 -1 . 2 0 0.23 5.32*
LMNT/GC 4.94* -1 . 2 2 2.82* 0.40 -3.88* -0 . 1 2
IR36/LMNT 5.41* -2.57* -4.52* -6.87** -5.96* 9.96**
IR36/ST 3.46* -0.44 -3.25* -4.56* 0.72 3.26*
IR36/GC 1.54* -1.42 -1.19 -1.76* -0 . 8 8 1.26*
ST/LMNT 5.13* -4.13* 2.04* 0.90 -2 .1 0 * -1.84*
ST/IR36 2.31* 0.79 1.84* 0.58 -0.37 -2.52*
ST/GC 3.87* 1.67* -4.88* -6 .0 2 ** 1.05 6.30**
GC/LMNT 3.65* -4.78* -0.26 -0.56 -2.13* 2.74*
GC/IR36 3.69* -0.10 -13.84** -14.56** -0.64 18.56**
GC/ST 3.25* -1.72* -8.55** -9.00** -1 . 1 0 13.36**
a m, mean of F 2 generation; a and d, pooled additive and dominance effects, respectively; aa, ad, and dd, pooled additive x additive, additive x dominance, and dominance x dominance effects, respectively.
k Estimates of m were always highly significant.
*, ** significant at 0.05 and 0.01 probability levels, respectively.
CHAPTER II
GENETICS OF CALLUS FORMATION RATE IN ANTHER CULTURE OF
RICE (ORYZA SATIVA L.)
ABSTRACT
A total of 375,873 rice anthers sampled from 10 parents,
the F^, F2 » and F 3 generations from a 4 x A diallel cross,
and 24 BCF^'s were inoculated on callus induction medium to
study the genetics of callus formation in rice anther
culture. Significant genotypic differences existed
among the 10 rice cultivars tested. The U.S. variety
'Lemont' had a high rate, 'Short Tetep' an intermediate, and
'IR36' and 'Gui Chow 1 low callus formation rates, and were
used as parents for a 4 x 4 diallel cross. Production of
callus by the 1 2 Fj hybrids was closely related to the low
parent, suggesting that suppression of callus formation is
dominant and high callus formation rates recessive. Maternal
effects did not generally appear important, with the
exception of a significant difference in F^ callus formation
rate for the reciprocal cross of Lemont and Short Tetep. The
mean callus formation rates of 24 BCF^'s tended to increase
when backcrossed to the high parent and decrease when
backcrossed to the low parent. Mean callus formation rate in
the 1 2 F2 *s was generally higher than the F 3 means.
Generation means analysis for 7 populations (Pj, P2 , Fj_, F 2 ,
46
47
F3 , and P2^1^ from 12 crosses revealed that dominance
and epistatic gene effects were of major importance in the
total variance, although additive gene effects appeared to be
important in some crosses. The heritability estimate
calculated by parent-offspring regression analysis is 0.33.
The results suggest that F 2 rather than F 3 material be
utilized for anther culture when crosses involve one parent
of high culturability and another of low.
Additional index words: diallel cross, generation mean
analysis, additive, dominance, epistatic, maternal effects,
heritability.
INTRODUCTION
In the last 20 years, considerable progress has been
made in applying anther culture techniques to varietal
improvement in rice (Chen, 1986a, b; Loo and Shu, 1986; Zhang
and Chu, 1986). Evidence accumulated since the first
reported success in anther culture of rice by Niizeki and
Oono (1968) indicates four major factors affecting anther
culture. These factors include the physiological status of
donor plants, anther pretreatments, culture conditions
(nutrient medium, temperature, etc.), and donor genotype.
Genotypic differences in callus induction and plant
48
regeneration rates in rice anther culture have been reported
in several studies (Niizeki and Oono, 1968; Mukherjee, 1973;
Chen et al., 1974; Lin et al., 1974; Oono, 1975; Chen and
Lin, 1976; Yin et al., 1976; Chen, 1978; Woo et al., 1978;
Woo and Huang, 1980; Cornejo-Martin and Primo-Millo, 1981;
Shen et al., 1982; Wakasa, 1982; Zhang and Chu, 1984; Chu et
al., 1984; Chu et al., 1985; Boyajiev and Kuong, 1986;
Davoyan, 1987). Genetic differences in culturability were
also found in stem node culture and immature panicle culture
of rice (Chu et al., 1986; Chu and Croughan, 1988). These
reports suggested that differences in culturability not only
existed among various rice species and subspecies such as
japonica, indica and javanica, but also between different
rice varieties.
Abe and Sasahara (1982) provided evidence for the
genetic control of culturability using cultivars of high and
low culturability and the F^ from their hybridization. Calli
induced from the F^ seeds showed a clear resemblance to the
parent of low culturability. They suggested that callus
formation were genetically recessive. Xui and Liu (1984)
analyzed the genetics of culturability by diallel analysis.
They found significant difference in general combining
ability (gca) and specific combining ability (sea) effects,
indicating additive and dominance gene effects which control
expression of culturability. Zhang and Chu (1985) and Chu
(1986) made a complete 5 x 5 diallel cross to study the
genetic background of callus formation and plant regeneration
49
in anther culture. The results indicated that maternal
effects, additive, and slightly high overdominance contribute
to genotypic differences. Miah et al. (1985) suggested that
culturability was inherited as recessive characters
controlled by a single block of genes.
The present study was conducted to evaluate genotypic
differences in callus formation rate among 9 indica varieties
and the U.S. variety Lemont; to clarify the role of maternal
effects in callus formation by comparing F^'s with their
reciprocals; to analyze the genetic background of the trait
through the evaluation of several generations, including F3 !s
and backcrosses to the F^'s; to investigate the contribution
of various gene effects by generation mean analysis; and to
calculate the heritability of the callus formation trait by
parent-offspring regression analysis.
MATERIALS AND METHODS
Varieties
The varieties used were Tetep, Lemont, Short Tetep,
Nanjing 11, Costa Rica 1119, Cica 6 , Gui Chow, IR36, IRGA
409, and Cica 8 . The origin of these varieties is presented
in Table 1.
Establishment of Progeny Populations
The varieties Lemont, IR36, Short Tetep, and Gui Chow
50
were identified as genotypes possessing differing rates of
callus formation and were used to produce a 4 x 4 diallel
cross. The hybridization method is described in Chapter 1.
Twelve F^'s were backcrossed to their corresponding parents
to produce P^F^ and P2 F 1 populations. F2 and F3 populations
were established by selfing.
Anther culture
Rice stems in the booting stage were collected on an
individual plant basis when the flag leaf collar was 2-5 cm
above the penultimate leaf collar. Following leaf blade
removal, the stems were sealed in plastic bags and given a
cold pretreatment of 5° C for 5-7 days. Before inoculation,
the rice boots were sterilized in a 50% bleach solution (v/v)
for 30 minutes. Panicles were aseptically dissected from the
stems and rinsed with distilled water three times. Based on
spikelet morphology, color of anthers, and the position of
the anthers within the spikelets, anthers at the uninucleate
stage of development were selected and plated onto N 6 medium
(Chu et al., 1975) in 50 mm x 10 mm petri dishes. Dishes
were sealed with Parafilm strips and placed in the dark at
25° C for callus induction.
A total of 32,806 anthers from ten varieties were
sampled from a complete randomized block design with 6 blocks
and 15 replications. A total of 100,500 anthers from 12 F2
populations, 66,700 anthers from 12 F 3 populations, and
114,050 anthers from 24 BCF^ populations were plated.
51
Callus Formation Rate
The number of anthers forming callus was cumulatively
recorded over a 70 day period following anther inoculation.
Calli were removed from each dish upon reaching 2 mm in
diameter. Callus formation rate was calculated as the number
of calli produced divided by the number of anthers inoculated
times 100 (see Chu et al., 1984).
Statistics
Generation mean analysis (Gamble, 1962) was used to
estimate genetic components of means and to evaluate the
contribution of various gene effects to the total variation
among generation means. The general model for the observed
mean of any generation, Y, is
Y = m + aa + Bd + a^aa + 2aBad + B^dd,
where m represents the mean of a reference population (F2 ); a
and d represent pooled additive and pooled dominance effects,
respectively; and aa, ad, and dd are the pooled digenic
interaction effects of additive x additive, additive x
dominance, and dominance x dominance gene effects,
respectively. The values of a and B are the genetic
components of the means of the 7 populations in this model
and are presented in Appendix 2, Table 1. Various gene
effects were calculated using the equations presented in
Appendix 2, Tables 2 and 3. Heritability estimates were
calculated based on parent-offspring regression analysis
(Smith and Kinman, 1965).
52
RESULTS AND DISCUSSION
I. Genotypic differences in callus formation rate of ten rice
varieties.
The range of callus formation rates for each individual
variety is presented in Table 1. Analysis of variance for
callus formation rate revealed a significant F value at the
0 . 0 1 probability level for genotypic differences among the
ten varieties. Comparison of the mean callus formation rate
suggested that the varieties can be classified as high,
intermediate, and low in callus induction (Table 1).
Significant differences observed in mean comparisons
(Duncan's multiple range test) among the ten cultivars
suggested that Tetep (11.84%) and Lemont (6.23%) have a high
callus formation rate and differ genotypically from the
intermediate group consisting of the varieties Short Tetep
(2.54%), Nanjing 11 (1.95%), Costa Rica (1.30%), and Cica 6
(1.14%). Callus formation rates for the varieties Gui Chow
(0.23%), IR36 (0.01%), IRGA 409 (0.01%), and Cica 8 (0.01%)
were significantly lower than rates in the other two groups.
II. Callus formation rate in a 4 x 4 diallel cross.
A complete diallel cross involving Lemont, IR36, Short
Tetep and Gui Chow was made to study the genetics of callus
formation. Lemont represents a high rate of callus
formation, Short Tetep an intermediate rate, and Gui Chow and
IR36 low rates. All Fj hybrids and their reciprocals
53
expressed callus formation rates resembling their low parents
except Short Tetep/Lemont (.13.02%, see Table 2). This
suggests that factors suppressive to callus formation are
dominant and expressed in the Fj generation. Comparison of
callus formation rates in Fj's and their corresponding
reciprocals reveals a significant difference for the cross
involving Lemont and Short Tetep, while the other 5 cross
combinations showed no significant difference in mean callus
formation rate for reciprocals (Table 2). Maternal effects
do not therefore generally appear important, but may be
significant for certain combinations of parents.
The observation in this study that F^ callus formation
rates resemble the lower parent in the cross are in general
agreement with previous findings with other varieties (Abe
and Sasahara, 1982; Xui and Liou, 1984; Zhang and Chu, 1985;
Miah et al., 1985; Chu and Zhang, 1985; Chu, 1986; Davoyan,
1987). The maternal effect observed in this study is similar
to the results reported by Zhang and Chu (1985).
III. Callus formation rates in BCF-^, F2 , and F3 progenies.
Callus formation rates in F2 's showed continuous
segregation (Table 3). Calculation of mean callus formation
rates in 1 2 F2 populations yielded higher values than their
corresponding Fj's, indicating that recombination increased
the F 2 mean value. The crosses having Lemont as one parent
generally produced more calli than other crosses. The F2 's
from Lemont/Short Tetep and Short Tetep/Lemont produced the
54
highest values in mean callus formation rate. Fg's from
Lemont/Gui Chow, IR36/Lemont, and Short Tetep/Lemont,
produced higher mean callus formation rates than other
crosses, suggesting a close association of high callus
formation rate with Lemont parentage (Table 4).
In general, the mean callus formation rate of BCF^'s
increased when backcrossed to the high parent, and decreased
when backcrossed to the low parent (Table 5). An example of
this pattern can be found in the crosses Lemont/IR36//Lemont
(4.56%) and in Lemont/IR36//IR36 (0.47%). The only
exceptions involved Lemont/ST//Lemont (8.22%), Lemont/ST//ST
(10.00%), ST/Lemont//ST (8.12%), and ST/Lemont//Lemont
(11.72%). This may relate to the maternal effect found in
the Lemont/Short Tetep as mentioned before. The increase
in mean value of these four BCF^'s may be attributable to
complex interactions between nuclear and cytoplasmic factors.
These findings regarding callus formation rates in
BCFj’s, F 2 !s, and F3 *s provide information of practical
significance for utilizing anther culture in rice varietal
improvement. When conducting anther culture of hybrids
derived from crossing two parents with high rates of callus
formation, anther culture of the F^ generation should produce
sufficiently high rates of callus formation. In the case
where the cross involved a low culturability parent, however,
callus formation rates are improved by utilizing the F 2
rather than F^ generation for anther culture.
55
IV. Generation means and estimates of gene effects.
The mean callus formation rates of 7 generations from 12
individual crosses are presented in Table 6 . In order to
analyze the genetic pattern for the trait in each cross, the
following examples are described in detail.
Lemont/IR36 : The mean callus formation rates for 7
generations (P3 , P2 » F^, ^ 2 * ^ 3 > **1^ 1 » an(* ^2 ^ 1 ̂ from
Lemont/IR36 are presented in Table 6 . The F^ value was close
to IR36. The F 2 mean was between the parents. The F 3 value
was similar to the F 3 value. The P^F^ value increased due to
backcrossing to the high parent Lemont, while the P2 F 1 value
decreased due to backcrossing to the low parent IR36.
Lemont/Short Tetep: The mean callus formation rates for
7 generations from Lemont/Short Tetep is shown in Table 6 .
The F^ value is statistically the same as the parent Short
Tetep. The F2 value was higher than both parents indicating
transgressive segregation. The F 3 value was close to the F 3
and Short Tetep. The Pi^i value increased due to
backcrossing to Lemont. However, the ?2^1 value also
increased when backcrossed to the intermediate parent Short
Tetep. This increase of mean value may be due to epistatic
gene effects.
Lemont/Gui Chow; Table 6 shows the mean callus
formation rate for 7 populations from Lemont/Gui Chow.
Again, the F 3 value (0.07) was close to the low parent Gui
Chow (0.23). The F 2 value was similar to the high parent
Lemont due to segregation for individuals with high values.
56
The mean F 3 value was 10.54 which showed a significant
increase from the Fj. The PIF^ value was close to the parent
Lemont and the value for the F2 generation due to the
backcrossing to Lemont, and the P2F^ value decreased due to
backcrossing to the low parent Gui Chow. The remaining
crosses followed the same genetic pattern with the exception
of IR36/Short Tetep and Short Tetep/Lemont (Table 6 ).
Based on the calculation of generation means for the
callus formation rate in the 1 2 crosses, estimates of six
parameters for gene effects are calculated and presented in
Table 7. The significant estimates are indicated at the 0.05
and 0 . 0 1 probability levels.
The contribution by additive gene effects (a) to
variation among the 1 2 crosses was statistically significant.
Five of 12 matings showed significant negative values and two
of 1 2 matings showed significant positive values.
Ten of 12 crosses showed significant values in dominance
gene effects (d) with negative values except for Short
Tetep/Lemont. Negative dominant effects can explain the F^
hybrids having low callus formation rates similar to their
low parents. The relative magnitude of dominance effects is
higher than the additive effects, suggesting that the
contribution of dominance is more important than that of
additive effects.
Additive x additive gene effects (aa) appear to be
important in 8 of 12 crosses. The magnitudes of aa are
relatively high in Lemont/Short Tetep, Lemont/Gui Chow, Gui
57
Chow/Lemont, and Gui Chow/IR36. Seven of 12 crosses showed
significant values of additive x dominance effects (ad).
Nine of 12 crosses showed significant values of dominance x
dominance effects (dd). Among epistatic effects, magnitudes
of dd appear to be higher than that of aa and ad, suggesting
a negative effect on the heritability of high callus
formation rates. The significance of aa, ad, and dd
indicates that epistatic gene effects play an important role
in the inheritance of callus formation rate. Generation
mean analysis of various gene effects on callus formation
rate revealed that the relative contributions of additive,
dominance, and epistatic gene effects to inheritance of the
trait are dependent on genotype. Dominance and dominance x
dominance effects contribute more, in general, to the total
variance, although additive effects are of importance for
some crosses.
Based on parent-offspring regression analysis,
calculation of the middle parent value to its F^ indicates a
low regression coefficient, yielding a heritability estimate
for callus formation rate of 0.33. Calculation of F2 mean
values to the F 3 values indicated a high regression
coefficient of 0.64, and the heritability estimate was 0.43.
Present data suggests that additive variance accounts, in
general, for a relatively small proportion of the overall
genetic variance.
58
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formation in rice. p. 419-420. In A. Fujiwara (ed.) Plant
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Boyajiev, P., and F.V. Kuong. 1986. Methods of inducing
callus formation and regeneration in anther culture of
rice. Selskostopanska Nauka 24 (2):92-97.
Chen, C.C. 1978. Effects of sucrose concentration on plant
production in anther culture of rice. Crop Sci. 18:905-
906.
Chen, C.C., and M.H. Lin. 1976. Induction of rice plantlets
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Chen, Y. 1986a. Anther and pollen culture of rice. p. 3-25.
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59
Chu, Q.R., and L.N. Zhang. 1985. Cytogenetics of aneuploids
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pollen grain culture of rice (Oryza sativa L.).
Euphytica 30:541-546.
Davoyan, E.I. 1987. Genetic determination of the process of
callus formation and induction of regenerates in the
tissue culture of rice. Genetika, USSR. 23 (2):303-310.
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60
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Bull. Natl. Inst. Agric. Sci. Ser. D26:139-222.
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offspring regression as an estimator of heritability.
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glaberrima Steud and its hybrids with 0. sativa L. Bot.
61
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China 2(suppl.):10-16.
62
Table 1. Callus formation rates of 10 varieties.
Variety Origin Number of
plates inoculated
Numberof
anthersinoculated
Mean(%)
Range of callus
formation rate (%)
Tetep Vietnam 19 954 11.84a1 9.72-12.89
Lemont U.S.A. 144 5,814 6.23a 5.14- 6.42
Short Tetep U.S.A. 34 1,734 2.54b 2.34- 2.66
Nanj ing 11 China 42 2 , 1 0 1 1.95bc 1.91- 2.03
Costa Rica U.S.A. 60 3,004 1.30bc 1.14- 1.52
Cica 6 Colombia 43 2,192 1.41bc 1.11- 1.58
Gui Chow China 109 5,452 0.23d 0.21- 0.24
IR36 IRRI 199 9,951 O.Old 0 .0 0 - 0 . 0 1
IRGA 409 Brazil 13 652 0. Old 0 .0 0 - 0 . 0 1
Cica 8 Colombia 19 952 O.Old 0 .0 0 - 0 . 0 1
* Means with the same letter are not significantly different at the 0.01 probability level according to Duncan's multiple range test.
63
Table 2. Callus formation rates of parents and their F ^ ’s.
Parents and F^'s N o . anthers inoculated
No. calli produced
Mean callus formation rate
(%)
Lemont 5,814 362 6.23
Lemont/IR36 2,602 19 0.73
Lemont/ST 2,901 41 1.41
Lemont/Gui Chow 2,957 2 0.07
IR36 9,951 1 0 . 0 1
IR36/Lemont 2,150 40 1 . 8 6
IR36/Short Tetep 1,951 14 0.72
IR36/Gui Chow 10,501 1 0 . 0 1
Short Tetep 1,734 44 2.54
ST/Lemont 3,180 414 13.02**
ST/IR36 2,550 5 0 . 2 0
St/Gui Chow 9,856 1 0 . 0 1
Gui Chow 5,452 13 0.23
Gui Chow/Lemont 3,356 3 0.09
Gui Chow/lR36 10,050 1 0 . 0 1
Gui Chow/ST 9,763 1 0 . 0 1
** significantly different from reciprocal cross at 0 . 0 1 probability level.
64
Table 3. Callus formation rates of F2 generation.
Cross No. of plates
inoculated
No. ofanthersplated
No. ofcalliproduced
Meancallus
formationrate(%)
Range(%)
Lemont/IR36 161 8,050 241 2.99 0 .00-15.60
Lemont/ST 117 5,850 630 10.77 0 . 22-39.09
Lemont/G 182 9,100 613 6.74 0 .00-20.44
IR36/Lemont 177 8,850 225 2.54 0 .00-10.83
IR36/ST 174 8,700 77 0.89 0 .00- 3.33
IR36/GC 153 7,650 28 0.37 0 .00- 1.14
ST/Lemont 206 10,300 981 9.52 0 .00-54.15
ST/IR36 2 1 0 10,500 79 0.75 0 .00- 5.64
ST/Gui Chow 247 7,350 92 1.25 0 .00- 3.45
GC/Lemont 198 9,900 92 0.93 0 .00- 4.44
GC/IR36 103 5,150 43 0.84 0 .00- 2.75
GC/ST 182 9,100 106 1.17 0 .00- 5.60
65
Table 4. Callus formation rates of E 3 generation.
Cross No. of No. of plates anthers
inoculated plated
No. ofcalliproduced
Meancallus
formationrate(%)
Range(%)
Lemont/IR36 92 4,600 27 0.59 0 .00- 3.00
Lemont/ST 97 4,850 1 2 0 2.47 0 .0 0 -1 1 . 0 0
Lemont/GC 128 6,400 685 10.55 1 .43-18.36
IR36/Lemont 129 6,450 362 5.61 0 .00-17.29
IR36/ST 159 7,950 2 2 0.28 0 .0 0 - 2 . 0 0
IR36/GC 106 5,300 1 0 0.19 0 .00- 0.33
ST/Lemont 1 0 2 5,100 500 9.80 1 .00-15.00
ST/IR36 62 3,100 7 0.23 0 .0 0 - 0.60
ST/Gui Chow 132 6,600 2 2 2 3.36 0 .0 0 -1 2 . 0 0
GC/Lemont 119 5,950 15 0.25 0 .0 0 - 1.08
GC/IR36 92 4,600 17 0.37 0 .00- 0.77
GC/ST 116 5,800 69 1.19 0 .00- 4.00
66
Table 5. Callus formation rates of 24 BCE^'s.
Backcross No. of No. of No. of Mean Rangeplates anthers
platedcalliformed
callus formation rate (%)
(%)
Lemont/IR36//Lemont 103 5,150 235 4.56 0.00-15.60Lemont/IR36//IR36 64 3,200 15 0.47 0.00- 0.91Lemont/ST//Lemont 302 15,100 1,241 8 . 2 2 0.37-14.27Lemont/ST//ST 7 350 35 1 0 . 0 0 4.00-16.00Lemont/GC//Lemont 109 5,450 361 6.42 0.17-20.80Lemont/GC//GC 103 5,150 137 2 . 6 6 0.18-10.00
IR36/Lemont//IR36 14 700 1 0 . 0 1 0 .0 0 - 0 . 1 0IR3 6 /Lemont//Lemont 1 1 0 5,500 275 5.00 0.89- 7.83IR36/ST//IR36 96 4,800 7 0.15 0.00- 0.50IR36/ST//ST 61 3,050 44 1.43 0.00- 3.00IR36/GC//IR36 67 3,350 1 1 0.33 0 .0 0 - 1 . 0 0IR36/GC//GC 94 4,700 2 1 0.45 0.00- 1.25
ST/Lemont//ST 1 0 2 5,100 414 8 . 1 2 1.60-22.44ST/Lemont//Lemont 268 13,400 1,571 11.72 5.33-22.33ST/IR36//ST 104 5,200 38 0.73 0.00- 1.78ST/IR36//IR36 45 2,250 24 1.07 0.00- 2.15ST/GC//ST 1 2 2 6 , 1 0 0 59 0.97 0.31- 2.00ST/GC//GC 103 5,150 41 0.80 0.00- 3.00
GC/Lemont//GC 84 4,200 7 0.17 0.00- 1.14GC/Lemont//Lemont 53 2,650 1 0 0.38 0 .0 0 - 1.82GC/IR36//GC 43 2,150 6 0.28 0.00- 0.50GC/IR36//IR36 2 2 1 , 1 0 0 3 0.27 0.00- 0.57GC/ST//GC 8 8 4,400 15 0.34 0 .0 0 - 1 . 1 1GC/ST//ST 117 5,850 130 2 . 2 2 0.00- 5.50
67
Table 6 . Generation mean for callus formation rates in anther culture of rice.
Cross Pj* ? 2 * 1 * 2 *3 PlFi *2 * 1
Lemont/IR36 6.23 0 . 1 0 0.73 2.99 0.59 4.56 0.47
Lemont/ST 6.23 2.54 1.41 10.77 2.47 8 . 2 2 1 0 . 0 0
LMNT/Gui Chow 6.23 0.23 0.07 6.74 10.54 6.62 2 . 6 6
IR36/Lemont 0 . 0 1 6.23 1 . 8 6 2.54 5.61 0 . 0 1 5.00
IR36/ST 0 . 0 1 2.54 0.72 0.89 0.23 0.15 1.44
IR36/Gui Chow 0 . 0 1 0.23 0 . 0 1 0.34 0.19 0.33 0.45
ST/Lemont 2.54 6.23 13.20 9.52 9.80 8 . 1 2 11.72
ST/IR36 2.54 0 . 0 1 0 . 2 0 0.75 0.23 0.73 1.07
ST/Gui Chow 2.54 0.23 0 . 0 1 1.25 3.36 0.97 0.80
Gui Chow/LMNT 0.23 6.23 0.09 0.93 0.25 0.17 0.38
Gui Chow/lR36 0.23 0 . 0 1 0 . 0 1 0.84 0.37 0.28 0.27
Gui Chow/ST 0.23 2.54 0 . 0 1 1.17 0.19 0.34 2 . 2 2
* P^ = female parent in cross = male parent in cross
i?i = pj/pjF2 = selfed seeds from F^F3 = selfed seeds from F2PfFi = F^ backcrossed to female parentP2 F 1 = F-̂ backcrossed to male parent
68
Table 7. Estimates of the genetic components of generation means for callus formation rates of 1 2 crosses.
gene effects
Cross ma »t> a d aa ad dd
LMNT/IR36 2.99 4.10** -4.30** -1.91** 0.98 -0.45
Lemont/ST 10.76 -1.78** -9.51** -6.64** -3.62** -18.20**
Lemont/GC 6.73 3.96** -11.53** -8.37** 0.96 -3.59**
IR36/LMNT 2.54 -4.98** -1.40* < i“IO1 -1.87** 0.07
IR36/ST 0 . 8 8 -1.29* -0.91* -0.36 -0.03 1.17**
IR36/GC 0.33 -0 . 1 1 0.09 0 .2 0 * -0.01 -1.49**
ST/Lemont 9.52 -3.60** 11.54** 1.58* -1.76* -6.09**
ST/IR36 0.75 -0.33 -0.47 0.58 -1.60** -1.23**
ST/GC 1.25 0.17 -2.58** -1.48** -0.98 0.74
GC/Lemont 0.92 -0 . 2 1 -5.76** -2.62** 2.79** 11.19**
GC/IR36 0.83 0 . 0 1 -3.61** -2.23** 1.16** 3.92**
GC/ST 1.16 -1 .8 8 ** 0.35 0.46 -1.99** -5.33**
a m, mean of F2 generation; a and d, pooled additive and dominance effects, respectively; aa, ad, and dd, pooled additive x additive, additive x dominance, and dominance x dominance effects, respectively.
k Estimates of m were always highly significant.
*,** Significant at 0.05 and 0.01 probability levels, respectively.
CHAPTER III
GENETICS OF PLANT REGENERATION IN ANTHER CULTURE OF RICE
(ORYZA SATIVA L.)
ABSTRACT
The genetics of plant regeneration from anther derived
callus was investigated using the 4 rice varieties 'Lemont',
'Short Tetep', 'IR36', and 'Gui Chow', the F^, F 2 , and F 3
generations from a diallel cross of these four varieties, and
24 BCF^'s. Green plant regeneration rates and albino
plantlet production rates were calculated as the number of
green and albino plants regenerated from 1 0 0 calli following
transfer to differentiation medium under uniform culture
conditions. Lemont regenerated plants at a rate of 3 plants
per 100 calli (3%), while IR36 and Gui Chow regenerated no
plants. The variety Short Tetep possessed a high
regeneration rate for both green plants (63%) as well as
albinos (316%). The rates for IR36 and Gui Chow were
significantly different from the rate for Lemont, which was
statistically different from the rate for Short Tetep. The
mean green plant and albino regeneration rates among the 1 2
F^'s showed overdominance and recessiveness in some crosses.
A significant reciprocal difference was found in the cross
involving Lemont and Short Tetep. Evaluation of the 24
BCF-^'s showed, in general, that the mean plant regeneration
69
70
rates increased upon backcrosseing to high parents and
decreased upon backcrossing to low parents. Generation mean
analysis indicates that dominance (d), additive x dominance
(ad), and dominance x dominance (dd) are the major
contributors to the variation of generation means, although
additive gene effect (a) are of importance in some crosses.
From a practical standpoint for rice anther culture,
relatively high regeneration rates can be obtained from a
cross involving a high and low parent by utilizing the F 2
generation for anther culture.
Additional index words: generation mean analysis, albino,
differentiation, additive, dominance, epistatic gene effects.
INTRODUCTION
In the last decade, rice anther culture research has
been shifting from basic studies on obtaining dji vitro
plantlets to applying this technique to rice varietal
improvement (Chen, 1986 a, b; Loo and Shu, 1986; Zhang and
Chu, 1986). Information accumulated since the first success
in rice anther culture reported by Niizeki and Oono (1968)
indicates the importance of genotypic effects in anther
culture. Anther culturability (callus formation rate x plant
regeneration rate) varies considerably between varieties.
Japonica types typically possess much higher culturability
than indica germplasm.
71
Cenol.yp ic differences in plant regeneration were Fi rst
reported by Niizeki and Oono (1968). Among the 10 japonica
rices they tested, only two cultivars produced plantlets.
Since then, similar findings have been reported by various
other research groups (Mukherjee, 1973; Chen et. al., 1974;
Lin et al., 1974; Oono, 1975; Chen and Lin, 1976; Yin et al.,
1976; Chen, 1978; Woo et al.,' 1978; Woo and Huang, 1980;
Cornejo-Martin and Primo-Millo, 1981; Abe and Sasahara, 1982;
Shen et al., 1982; Wakasa, 1982; Zhang and Chu, 1984;
Croughan et al., 1984; Chu et al., 1984; Chu et al., 1985;
Croughan et al., 1985; Miah et al., 1985; Boyajiev and Kuong,
1986; Chu et al., 1986; Croughan et al., 1986; Davoyan,
1987). These reports revealed that plant regeneration not
only differed among various wild species of rice and
subspecies such as japonica, indica and javanica, but also
between cultivars of rice.
Xui and Liu (1984) analyzed the genetic aspects of
culturability by diallel analysis. They found significant
differences in general combining ability (gca) and specific
combining ability (sea) effects, indicating that additive and
dominance gene effects were important aspects of
culturability. Zhang and Chu (1985) and Chu (1986) made a
complete 5 x 5 diallel cross to study the genetics of plant
regeneration. The results indicated that maternal, additive,
and slightly high overdominance effects contributed to the
observed genotypic differences.
The present study was conducted to systematically
72
evaluate the genetics underlying plant regeneration in rice
anther culture. Specifically, the objectives were to clarify
the role of maternal effects in plant regeneration by
comparing F 3 hybrids and their reciprocals; to analyze the
genetic aspects of the trait through analysis of the P-̂ , P2 ,
Fi* F2 , F 3 , PjFj, and P2 F 1 generations; to investigate the
contribution of various gene effects by generation mean
analysis, and to calculate the heritability of the character
by parent-offspring regression analysis.
MATERIALS AND METHODS
Establishment of experimental material
The varieties 'Lemont1, 'IR36', 'Short Tetep', and 'Gui
Chow 1 were selected from a screening of ten varieties to
represent high and low culturability parents for use in a 4 x
4 diallel cross. The details of the hybridization method are
presented in Chapter One. Twelve F^'s were backcrossed to
their corresponding parents to produce and ^2 ^ 1
populations (Appendix 1, Table 2). F2 and F3 populations
were established by selfing.
Plant regeneration
Anthers of parents, 12 F^'s, 24 BCF^'s, 12 F2 's, and 12
F3 's were cultured on N 6 medium to induce callus formation.
Details of callus induction were presented in Chapter Two.
Callus which formed was transferred to regeneration medium
73
upon reaching 2 mm in diameter. Regeneration medium was MS
medium supplemented with 0.5 mg/1 NAA and 2.0 mg/1 KT. The
cultures were maintained at 25° C with 16:8 hours of
florescent light. Upon reaching the 3-leaf stage,
regenerated plantlets were subcultured to fresh dishes of the
same medium to promote continued development.
Statistics
The plant regeneration rate was expressed as a
percentage calculated as the number of green plantlets
obtained divided the by number of calli transferred.
Generation mean analysis (Gamble, 1962) was used to estimate
genetic components of means for green plant and albino plant
regeneration rates. To evaluate the contribution of various
gene effects to the total variation among generation means, a
six parameter model was used to calculate estimates of
genetic effects. The general model for the observed mean of
any generation, Y, is
Y = m + aa + Bd + a^aa + 2aBad + B^dd,
where m represents the mean of a reference population a
and d represent pooled additive and pooled dominance effects,
respectively; and aa, ad, and dd are the pooled digenic
interaction effects of additive x additive, additive x
dominance, and dominance x dominance gene effects,
respectively. The value of a and B for the genetic
components of the means of the 7 populations are listed in
Appendix 2, Table 1. The equations for calculation of
74
various gene effects are presented in Appendix 2, Tables 2
and 3.
RESULTS AND DISCUSSION
I. Green and albino plant regeneration rates for parents and
Fx 1 s.
Green and albino plant regeneration rates are presented
in Table 1. The variety Short Tetep had the highest value
for green plant regeneration (64%). Lemont had an
intermediate plant regeneration rate (3%), while IR36 and
Gui Chow produced no plantlets. Significant differences in
plant regeneration rate were found in mean comparisons
between Short Tetep and Lemont, and between Lemont and IR36.
The mean plant regeneration rates in the 12 F^'s ranged from
0 to 2 0 0 % and significant differences were found among F^'s
and their reciprocals. This indicates that maternal effects
may play a role in plant regeneration. All crosses having
Lemont as one parent produced green plants except Lemont/Gui
Chow. The mean plant regeneration rates in the crosses
Lemont/lR36, IR36/Lemont, Short Tetep/Lemont, and Gui
Chow/Lemont exceeded both parents, indicating the occurence
of overdominance. Four crosses, IR36/Gui Chow, Short
Tetep/Gui Chow, Gui Chow/lR36, and Gui Chow/Short Tetep,
failed to produce callus and therefore regenerated no plants.
Regarding albino plant regeneration rates, Short Tetep was
75
the highest among the four varieties. Significant
differences existed among varieties and their F^'s for this
trait.
The mean regeneration rates of the four parents and
their F^'s in this study showed overdominance and maternal
effects in some crosses, similar to the results in an earlier
report (Zhang and Chu, 1985). However, high plant
regeneration rates appeared recessive in the crosses
Lemont/Short Tetep, Lemont/Gui Chow, IR36/Short Tetep,
IR36/Gui Chow, Short Tetep/IR36, Short Tetep/Gui Chow, Gui
Chow/Short Tetep, and Gui Chow/lR36. The repression of plant
regeneration in these Fj crosses may be attributable to the
parents Gui Chow and IR36, which possess very low
regeneration capabilities.
II. Green and albino plant regeneration rates in F2 and F 3
populations.
A total of 3,207 calli from 12 F2 populations were
transferred to differentiation medium to evaluate plant
regeneration rates (Table 2). No plants were regenerated
from the cross IR36/Gui Chow and its reciprocal, reflecting
the poor regeneration rates of both parents. The other 10
crosses varied in their mean regeneration rates, ranging from
2.5% for Short Tetep/IR36 to 121% for Short Tetep/Lemont.
Genotypic differences were found to be significant among
F2 's. All F 2 's produced various amount of albinos, ranging
76
from 13% for Lemont/IR36 to 360% for Gui Chow/Lemont.
Green plant and albino regeneration rates for the 12 F3
populations are shown in Table 3. The mean green plant
regeneration rate varied from 0% for IR36/Gui Chow to 335%
for Gui Chow/lR36. The albino regeneration rates of the 12
F 3 's ranged from 0% to 311%.
Green plant and albino regeneration rates in the F2 and
F3 generations followed a continuous segregation pattern.
However, generation means of the traits shifted for
individual crosses. The genetic factors involved in
regeneration from anther culture therefore appear more
complex than those involved in immature panicle culture
(Chapter One) and in the formation of callus from anthers
(Chapter Two).
III. Green plant and albino regeneration rates in 24 BCF^'s.
The mean green plant and albino regeneration rates of 24
BCF^'s are presented in Table 4. The mean green plant
regeneration rates of BCF^'s increased, in general, when the
F 3 was backcrossed to the high parent, and decreased when it
was backcrossed to the low. An example can be found in the
BCF3 of Lemont/IR36//Lemont (9.8%) and in Lemont/lR36//lR36
(0%). However, BCF^'s of Lemont/Gui Chow//Lemont, Lemont/Gui
Chow//Gui Chow, Gui Chow/Short Tetep//Gui Chow and Gui
Chow/Short Tetep//Short Tetep did not follow this general
trend, possibly due to some unique genetic aspect of the Gui
Chow genotype.
77
Albino rates in BCF^'s were dependent on the individual
cross. Most crosses which involved Short Tetep as a parent
produced high rates of albinos. The trait of producing
albino regenerates appears highly heritable, since Short
Tetep had the highest albino production rate (316%) among the
4 parents.
IV. Genetic estimates of green plant and albino regeneration
rates.
Mean plant regeneration rates are shown in Table 5.
Based on these data, the genetic estimates of additive (a),
dominance (d), additive x additive (aa), additive x dominance
(ad), and dominance x dominance (dd) were calculated (Table
6 ). Additive (a) gene effects affected plant regeneration
rates in most crosses except Lemont/Gui Chow, IR36/Gui Chow,
and Gui Chow/lR36. In the case of IR36/Gui Chow and its
reciprocal cross, both parents produced no regenerants and
genetic estimates could not be made. However, the relative
magnitude of additive gene effects to the mean effects (m) is
small compared to dominance effects (d). Therefore, it
contributes less to the total variation of the generation
means. On the other hand, dominance (d), additive x additive
(aa), and dominance x dominance (dd) gene effects showed
highly significant values, indicating that these factors are
significant contributors to the variation of the means.
The mean albino plant regeneration rates are shown in
Table 7. The genetic estimates calculated by generation mean
78
analysis are presented in Table 8 . Additive gene effects (a)
appear to be significant in 8 of 1 2 crosses with relatively
small magnitudes for the mean effects (m). However,
dominance (d), additive x additive (aa), and dominance x
dominance (dd) are significant with relatively large
magnitudes to their mean effects. Therefore, these gene
effects play an important role in the total variation of the
generation means.
The present study on green and albino plant regeneration
revealed a complicated genetic background controlling these
traits. Both overdominance and recessiveness were observed
among the various crosses. In addition, significant maternal
effects were observed. It is suggested that expression of
high regeneration rates may involve the interaction of
cytoplasmic factors and nuclear genes. Significant values of
dominance gene effects (d), epistatic effects (aa, ad, and
dd) may contribute more to the variance of generation means
than additive effects (a).
79
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83
Table 1. Plant regeneration in 4 parents and their 12 F-^'s.
Parents No. calli and transferred F i ’s
No.green
plantletsproduced
No.albino
plantletsproduced
Mean green regen. rate(%)
Meanalbinoregen.rate(%)
Lemont 362 11 53 3.0 14.6
Lemont/IR36 19 16 5 84.2 26.3
Lemont/ST 41 5 42 1 2 . 2 102.4
Lemont/GC 2 0 0 0 . 0 0 . 0
IR36 1 0 1 0 . 0 1 0 0 . 0
IR36/Lemont 40 11 14 27.5 35.0
IR36/ST 14 0 3 0 . 0 21.4
Short Tetep 44 28 139 63.6 315.9
ST/Lemont 414 830 419 200.4 1 0 1 . 2
ST/IR36 5 0 18 0 . 0 360.0
Gui Chow 13 0 8 0 . 0 61.5
GC/Lemont 3 3 3 1 0 0 . 0 1 0 0 . 0
84
Table 2. Green and albino plant regeneration rates in F2's ’
Cross No. calli No.transferred green
plantletsproduced
Lemont/lR36 241 37
Lemont/ST 630 624
Lemont/GC 613 576
IR36/Lemont 225 24
IR36/ST 77 53
IR36/GC 28 0
ST/Lemont 981 1,191
ST/IR36 79 2
ST/GC 92 29
GC/Lemont 92 75
GC/IR36 43 0
GC/ST 106 31
N o . Mean Meanalbino green albino
plantlets regen. regen.produced rate(%) rate(%)
32 15.3 13.2
543 99.0 8 6 . 1
303 93.9 9.4
92 1 0 . 6 40.8
186 6 8 . 8 241.5
17 0 . 0 60.7
1,393 121.4 142.0
137 2.5 173.4
235 31.5 255.4
331 81.5 359.7
115 0 . 0 267.4
339 29.2 319.8
85
Table 3. Plant regeneration in F3 generation.
Cross No. calli No. No. Meantransferred green albino green
plantlets plantlets regen.produced produced rate(%)
Lemont/IR36 27 0 0 0
Lemont/ST 1 2 0 147 229 122.5
Lemont/GC 685 252 311 36.7
IR36/Lemont 362 75 78 20.7
IR36/ST 2 2 39 13 177.2
IR36/GC 1 0 0 0 0 . 0
ST/Lemont 500 414 350 82.8
ST/IR36 7 16 9 228.5
ST/GC 2 2 2 72 691 32.4
GC/Lemont 15 42 18 280.0
GC/IR36 17 57 16 335.2
GC/ST 69 5 127 7.2
Mean albino regen. rate(%)
0190.8
45.A
21.5
59.0
0.070.0
128.5
311.2
128.5
94.1
184.0
Table 4. Regeneration rates of 24 BCFj's.
Cross No. calli transferred
N o . N o . Mean Meangreen albino green albino
plantlets plantlets regen. regen.produced produced rate(%) rate(%)
Lemont/IR3 6 //Lemont 235 23 18 9.7 7.6Lemont/IR36//IR36 15 0 0 0 . 0 0 . 0Lemont/ST//Lemont 1,241 732 940 58.9 75.7Lemont/ST//ST 35 117 99 334.2 282.8Lemont/GC//Lemont 361 84 143 23.2 39.6Lemont/GC//GC 137 32 91 23.3 66.4
IR36/Lemont//lR36 53 2 4 3.7 7.5IR36/Lemont//Lemont 275 17 36 6 . 1 13.0IR36/ST//IR36 7 0 6 0 . 0 85.7IR36/ST//ST 44 8 94 18.1 213.6IR36/GC//IR36 11 0 4 0 . 0 36.3IR36/GC//GC 2 1 0 14 0 . 0 6 6 . 6
ST/Lemont//ST 1,571 1,149 1,132 73.1 72.0ST/Lemont//Lemont 414 458 538 1 1 0 . 6 129.9ST/IR36//ST 38 28 42 73.6 110.5ST/IR36//IR36 24 17 15 70.8 62.5ST/GC//ST 59 47 138 79.6 233.9ST/GC//GC 41 15 1 1 2 36.5 273.1
GC/Lemont//GC 7 0 14 0 . 0 2 0 0 . 0GC/Lemont//Lemont 1 0 11 2 2 1 1 0 . 0 2 2 0 . 0GC/IR36//GC 6 0 0 0 . 0 0 . 0GC/IR36//IR36 3 0 0 0 . 0 0 . 0GC/ST//GC 15 63 13 420.0 8 6 . 6GC/ST//ST 130 24 257 18.4 197.6
87
Table 5. Mean plant regeneration rates for parents and progeny.
Cross P 1 ? 2 Fl f 2 *3 PlFi P2 F 1
LMNT/IR36 3.0 0 . 0 84.2 15.3 0 . 0 9.8 0 . 0
LMNT/ST 3.0 63.6 1 2 . 2 99.0 122.5 58.9 334.2
Lemont/GC 3.0 0 . 0 0 . 0 93.9 36.7 23.2 23.3
IR36/LMNT 0 . 0 3.0 27.5 1 0 . 6 20.7 3.7 6 . 1
IR36/ST 0 . 0 63.6 0 . 0 6 8 . 8 177.2 0 . 0 18.1
IR36/GC 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0
ST/LMNT 63.6 3.0 200.4 121.4 82.8 73.1 1 1 0 . 6
ST/IR36 63.6 0 . 0 0 . 0 2.5 228.5 73.6 70.8
ST/GC 63.6 OO
0 . 0 31.5 32.4 79.6 36.5
GC/LMNT 0 . 0 3.0 1 0 0 . 0 81.5 280.0 O•O
1 1 0 . 0
GC/IR36 0 . 0 0 . 0 0 . 0 0 . 0 335.2 0 . 0 0 . 0
GC/ST 0 . 0 63.6 0 . 0 29.2 7.2 420.0 18.4
* P-̂ = female parent in cross P2 = male parent in cross Fl - P!/P2F 2 - selfed seeds from F^F 3 = selfed seeds from F2PlFi = F^ backcrossed to female parentP2 F 1 = F^ backcrossed to male parent
88
Table 6 . Genetic components of plant regeneration rates of parents and progeny.
Cross m a d aa ad dd
1 15.3 9.8* 41.0** -41.6** 8.3* 193.3**
2 99.0 -275.3** 401.0** 390.3** -245.01** -1,085.8**
3 93.9 -0 . 1 -284.1** -282.5** -1 . 6 192.3**
4 1 0 . 6 -2.4* 3.2* -22.7** -0.9 60.8**
5 6 8 . 8 -18.1* -270.7** -238.9** 13.6* -266.2**
6 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0
7 121.4 -37.4* 49.0* -118.1** -67.7* 218.2**
8 2.5 2 .8 * 247.0** 278.9** -28.9** -504.2**
9 31.5 43.0** 74.6** 106.4** 1 1 .2 * -275.2**
1 0 81.5 -1 1 0 ,0 ** -7.6 -106.0** -108.4** 89.1**
11 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0
1 2 29.2 401.3** 728.1** 759.9** 443.3** -1,573.2**
a m, mean of F2 generation; a and d, pooled additive and dominance effects, respectively; aa, ad, and dd, pooled additive x additive, additive x dominance, and dominance x dominance effects, respectively.
k Estimates of m were always highly significant.
significant at 0.05 and 0.01 probability levels, respectively.
Cross 1: Lemont/lR36, 2: Lemont/ST, 3: Lemont/Gui Chow, 4: IR36/Lemont, 5: IR36/ST, 6 : IR36/Gui Chow, 7: ST/Lemont, 8 : ST/IR36, 9: ST/Gui Chow, 10: Gui Chow/Lemont, 11: Gui Chow/lR36, and 12: Gui Chow/ST.
89
Table 7. Mean albino plant regeneration rate for parents and progeny.
Cross P 1 p 2 F 1 * 2 f 3 PlFi p2 pl
LMNT/IR36 14.6 1 0 0 . 0 26.3 3.2 0 . 0 7.6 0 . 0
Lemont/ST 14.6 315.9 102.4 8 6 . 1 190.8 75.7 282.8
Lemont/GC 14.6 0 . 0 0 . 0 49.4 45.4 39.6 66.4
IR36/LMNT 1 0 0 . 0 14.6 35.0 40.8 21.5 7.5 13.0
IR36/ST 1 0 0 . 0 315.9 21.4 241.5 59.0 85.7 213.6
IR36/GC 1 0 0 . 0 0 . 0 0 . 0 60.7 0 . 0 36.3 6 6 . 6
ST/LMNT 315.9 14.6 124.3 142.0 70.0 72.0 129.9
ST/IR36 315.9 1 0 0 . 0 360.0 173.4 128.5 110.5 62.5
ST/GC 315.9 0 . 0 0 . 0 255.4 311.2 233.9 273.1
GC/LMNT 0 . 0 14.6 1 0 0 . 0 359.7 1 2 0 . 0 2 0 0 . 0 2 2 0 . 0
GC/IR36 0 . 0 1 0 0 . 0 0 . 0 267.4 94.1 0 . 0 0 . 0
GC/ST 0 . 0 315.9 0 . 0 319.8 184.0 8 6 . 6 197.6
* Pj = female parent in cross P2 = male parent in cross*1 =F 2 = selfed seeds from F 3F 3 = selfed seeds from F2PfFi = Fj backcrossed to female parentP2F 3 = F^ backcrossed to male parent
90
Table 8 . Genetic components of albino regeneration rates.
Cross ma ,b a d aa ad dd
1 13.2 7.6* -68.7** -37.7** 50.3** 189.7**
2 8 6 . 1 -207.1** 309.6** 372.4** -56.4** -554.2**
3 49.4 -26.8* 7.0 14.3 -34.1* -211.7**
4 40.8 -5.5 -144.6** -1 2 2 .2 ** -48.2* 256.6**
5 241.5 -127.9** -554.0** -367.5** -19.9 227.6**
6 60.7 -30.3** -86.7** -36.7** -80.3** -69.2**
7 142.0 -57.8* -204.9** -163.9** -2 0 . 8 339.1**
8 173.4 40.8* -159.5** -347.6** -59.9* 1,137.4**
9 255.4 39.2 -165.5** -7.5 -197.2** -690.6**
1 0 359.7 -2 0 . 0 -506.4** -559.1** -1 2 . 6 -26.2
11 267.4 0 . 0 -1,119.7** -1,069.7** 50.0 1,169.7**
1 2 319.8 -1 1 2 .0 * -868.4** -710.5** 46.9 457.7**
a m, mean of F2 generation; a and d, pooled additive and dominance effects, respectively; aa, ad, and dd, pooled additive x additive, additive x dominance, and dominance x dominance effects, respectively.
b Estimates of m were always highly significant.
*,** significant at 0.05 and 0.01 probability levels, respectively.
Cross numbers are the same as in Table 6 .
SUMMARY
A comprehensive study was made on the genetics of callus
formation and plant regeneration rates in both anther culture
and immature panicle culture of rice (Oryza sativa L.). Ten
rice varieties including Lemont, Tetep, Short Tetep, IR36,
Gui Chow, Nanjing 11, Cica 6 , Cica 8 , Costa Rica 1113, and
IRGA 409 were used as preliminary experimental materials to
study genotypic differences in culturability in vitro. Four
varieties, Lemont, IR36, Gui Chow, and Short Tetep, were then
selected as representing high, intermediate, and low
culturability parents to establish a 4 x A diallel cross.
Backcrosses were made and corresponding F2 and F3 populations
were developed by selfing.
A total of 6,332 rice immature panicles derived from
seven populations including parents (P^ and P2 )> Fi's, F2 *s,
and F 3 's from 12 crosses were cultured in vitro. Significant
genotypic differences in regeneration rate were found among
the ten varieties. The variety Lemont possessed a high
regeneration rate, averaging 6 . 6 plants per panicle, and its
descending progenies (BCF^, F 2 , and F 3 ) inherited this
character. In contrast, the indica rice variety IR36 and its
progeny had low regeneration rates. Generation mean analysis
of regeneration revealed significant values for dominance
gene effects (d) and epistatic effects (additive x additive,
additive x dominance, and dominance x dominance). The
91
92
heritability estimate (h) calculated by parent-offspring
regression was 0.63, indicating that this trait is highly
heritable.
A total of 375,873 rice anthers sampled from the above
populations were inoculated onto callus induction medium.
Genotypic differences were significant among cultivars.
Production of callus by 12 F-̂ hybrids was closely related to
the low parent, suggesting that factors suppressive to callus
formation are dominant and that high callus formation rates
are recessive. A significant difference in callus formation
rate was observed for Lemont/Short Tetep and its reciprocal
cross, indicating maternal effects. Evaluation of the mean
callus formation rates of 24 BCF^'s showed that rates
increased upon backcrossing to the high parents and decreased
upon backcrossing to the low parents. The mean callus
formation rates of the 1 2 F 2 *s were generally higher than the
F^ means. Generation mean analysis of the 7 populations
evaluated for each of the 1 2 crosses revealed that dominance
and epistatic gene effects are of major importance to the
total variance, although additive gene effects appear to be
important in some crosses. The heritability estimate
calculated by parent-offspring regression analysis is 0.34.
Green and albino plant regeneration rates, expressed as
a percentage calculated as the number of plants regenerated
from 1 0 0 calli transferred to regeneration medium, was
evaluated for anther culture. The variety Short Tetep
produced high rates of regeneration of both green plants
93
(63%) and albinos (316%). Lemont was an intermediate type in
green plant regeneration (3%), while IR36 and Gui Chow
produced no plants. A statistically significant difference
in regeneration was obtained among the 4 genotypes tested.
The mean green plant and albino regeneration rates of the 12
F^'s showed overdominance and recessiveness in some crosses
and a significant reciprocal difference was found for
Lemont/Short Tetep. Twenty four BCF^'s showed, in general,
that the mean plant regeneration rate increased when
backcrossed to the high parents and decreased when
backcrossed to the low parents. Generation mean analysis
indicated that dominance (d), additive x dominance (ad), and
dominance x dominance (dd) are important contributors to the
variation of generation means, although additive gene effects
(a) were important in some crosses. It is suggested that F 2
rather than F-̂ material be utilized for anther culture when
the crosses involve one high and one low culturability
parent.
Genetic analysis in the study indicated that the
genetic background of the callus formation and plant
regeneration rates differ from immature panicle culture and
anther culture. High regeneration rates appeared to be
dominant in immature panicle culture while recessive in
anther culture. Significant epistatic gene effects in callus
formation and plant regeneration rates found in the study
suggest that recurrent selection may be an effective method
for improving these traits.
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Shanghai 1(3):1-10.
Zhang, Z.H., and Q.R. Chu. 1986. Advances in rice anther
culture for varietal improvement in China. J. Agric.
Sci. China 2 (Suppl.):10-16. In. Proc. Sino-Jpn. Symp.
Biotech.
Zhao, C.Z., K.L. Zheng, X.F. Qi, Z.X. Sun, and Y.P. Fu. 1982.
Characteristics of rice plants derived from somatic
tissue and their progenies in paddy fields. Acta Genet.
Sin. 9:320- 324.
Zheng, Z.L., and Q.R. Chu. 1984. Selection of mutant cell
line resistant to raw toxin of blast in rice. Bot. Bull.
Sin. 11:24-33.
Zheng, Z.L., Q.R. Chu, and C.M. Zhang. 1985. Pathogenecity
of culture filtrate from blast pathogen on rice anther
culture. Acta Agric. Shanghai l(2):85-90.
Zhou, G.Z., M.M. Yei, and Y.F. Jia. 1986. Establishment of
rice cell line and plantlets regeneration from single
cell culture. Acta Agric. Shanghai 2(3):1-8.
Zhu, D.Y., and C.C. Wang. 1982. Effect of the preliminary
centrifugal treatment on the pollen induction in rice.
Acta Biol. Exp. Sin. 15:127-130.
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Zimmy, J., and H. Lorz. 1986. Plant regeneration and
initiation of cell suspensions from root-tip derived
callus of Oryza sativa L. (rice). Plant Cell Rep. 5:89-
92.
APPENDIX I
RICE VARIETAL IMPROVEMENT THROUGH ANTHER CULTURE
AND IMMATURE PANICLE CULTURE
In the studies on the genetics of callus formation and
plant regeneration in rice, a A x A diallel cross was made
involving Lemont, Short Tetep, IR36, and Gui Chow. The
vacuum pump emasculation and hand pollination yielded 8 A
crosses with 1,070 F^ seeds (Table 1). BCFj populations were
established by backcrossing 1 2 F^'s to their corresponding
parents, yielding 83 crosses with 1,589 backcrossed seeds
(Table 2).
A total of 6,332 immature panicles from the 10 parental
varieties, 12 F^'s, 12 F2 's, 12 F3 *s and 2A BCF^'s were
plated on RA medium in 1986 and 1987, producing 28,398
regenerated plants (Table 3). A total of 375,873 anthers
from these materials were inoculated (Chapter 2), and
produced 15,003 regenerated plants (Table 3).
A total of 10,582 regenerated lines from both anther and
immature panicle culture were grown in 1987 (Table A). A
total of A27 rows and single plants were selected among and
within the progeny populations derived from in vitro culture
for further tests in 1988. These selected lines are being
evaluated for useful germplasm for Louisiana's varietal
improvement program.
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Table 1. A 4 x A diallel cross made in 1986.
Cross Number of crosses Number of hybridmade seeds obtained
Lemont/lR36 7 70
Lemont/Short Tetep 5 55
Lemont/Gui Chow 1 0 50
IR36/Lemont 6 37
IR36/Short Tetep 4 40
IR36/Gui Chow 6 45
Short Tetep/Lemont 7 58
Short Tetep/IR36 7 1 0 1
Short Tetep/Gui Chow 15 234
Gui Chow/Lemont 2 41
Gui Chow/lR36 1 2 260
Gui Chow/Short Tetep 3 79
Total 84 1,070
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Table 2. Backcrosses made in summer of 1986.
Backcross Number of Number ofcrosses made seeds obtained
Lemont/IR36//Lemont Lemont/IR36//IR36 Lemont/ST//Lemont Lemont/ST//ST Lemont/Gui Chow//Lemont Lemont/Gui Chow//GuiChow
IR36/Lemont//IR36IR36/Lemont//lR36IR36/ST//IR36IR36/ST//STIR36/GuiChow//lR36IR36/Gui Chow//GuiChow
ST/Lemont//STST/Lemont//LemontST/IR36//STST/IR36//IR36ST/Gui Chow//STST/Gui Chow//Gui Chow
GuiChow/Lemont//GC Gu i Chow/Lemont//Lemont Gui Chow/lR36//GC Gui Chow/lR36//IR36 Gui Chow/ST//Gui Chow Gui Chow/ST//ST
3 375 443 434 1034 994 126
2 2 22 263 464 794 1323 106
5 904 453 512 703 643 50
3 584 263 383 913 616 82
Total 83 1,589
Table 3. Plant regeneration from anther culture and immature panicle culture.
Year Material Explant Number oftype regenerants
1986 10 varieties, panicle 9,074
12 F^ hybrids anther 1,589
1987 12 F x, 12 F2 , panicle 19,324
12 F 3 , 24 BCF^ anther 13,414
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Table 4. Regenerated lines evaluated in the field in 1987.
Variety Explant type Number of lines
Lemont immature panicle 5,978
anther 874
Tetep immature panicle 2 1 1
anther 11
Gui Chow immature panicle 627
Nanjin 11 immature panicle 1 0 1
Short Tetep immature panicle 268
anther 14
Costa Rica immature panicle 71
IR36 immature panicle 2 0
Cica 6 immature panicle 19
IRGA 409 immature panicle 147
Fi's immature panicle 1,576
anther 665
APPENDIX 2
GENERATION MEAN ANALYSIS: MODEL AND FORMULA
Table 1. Coefficients for genetic components of means of each of seven populations.
Generations
Component
m a d aa ad dd
Parent 1 (Pj) 1 1 -0.5 1 - 1 0.25
Parent 2 (P2 ) 1 -1 -0.5 1 - 1 0.25
F 1 1 0 0.5 0 0 0.25
F 2 1 0 0 0 0 0
f3 1 0 -0.25 0 0 0.625
P 1 F 1 1 0.5 0 0.25 0 0
P2 f 1 1 -0.5 0 0.25 0 0
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Table 2. Equations used for calculation of parameters.
Parameter Equation
m F2
a = ^ 1 ^ 1 “ ^2 ^ 1
d = -0.5 P^ - 0.5 Pj + fJ - 4 F^ + 2 ?1fl + 2 P p 7
aa — - 4 F 2 + 2 P^F^ + 2 P2 F^
ad = -0.5 + 0.5 P2 + PiFi + ^2^1
dd — Pj + P2 + 2 F^ + 4 F2 - 4 P^F^ -4 P2F 1
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Table 3. Equations for calculating variance of estimates.
Variance Equation
sm2 = sF2
sa2 = sPjFi2 + s P p T 2
sd2 = 0.25sP^2 + 0.25sP2^ + sF^2+16sF2^ + 4sP^F^ 2 + AsP^F^ 2
saa2= 1 6 sF2 ^ + AsP^F^ 2 + AsP^F^ 2
sad - 0.253?^ + 0.25sP2^ sPlF l + sP2*Y
sdd2= sPj2 + sP2 2 +AsF12+16sF22+ lasP^ 2 +16sP2F]/
VITA
Qi Ren Chu was born on August 5th, 1950 in Shanghai, the
People's Republic of China. He entered primary and high
school in his hometown and attended the Shanghai Normal
University in 1973. He received a Bachelor of Science degree
in Biology in 1977. After his graduation, he was assigned to
the Shanghai Academy of Agricultural Sciences as a research
assistant in rice breeding. In 1979, he advanced to the
position of research associate at the institute. He received
a scholarship from the International Rice Research Institute
and entered the University of The Philippines at Los Banos in
1980. He received his Master of Science degree in Agronomy
under the direction of the IRRI rice geneticist, Dr. T. T.
Chang.
Upon receipt of his M.S., Qi Ren Chu returned to China
and served at the Shanghai Academy for an additional four
years, during which he became an assistant scientist, project
learder, associate plant breeder, and head of the Tissue
Culture Laboratry. He has authored 31 publications and
translated two textbooks into Chinese. He has received
awards for scientific achievement from both the national and
Shanghai Municipal governments. In 1986, he received a
graduate research assistantship from the L.S.U. Agricultural
Center to support his doctoral studies in the Department of
Agronomy and at the Rice Research Station. He is presently a
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candidate for the degree of Ph.D.
Qi Ren Chu is married to Xin Hua Wang and has one son, Yi
Jia Chu.
DOCTORAL EXAMINATION AND DISSERTATION REPORT
Candidate: Qi Ren Chu
Major Field: Agronomy
Title of Dissertation: G e n e t ic s o f C a llu s F orm ation and P la n t R e g e n e r a tio n in R ice( Oryza s a t i v a L . )
Approved:
Major Professor and Chairman
Dean of the Graduate School
EXAMINING COMMITTEE:
Date of Examination:
April 14, 1988