Planta (1985)165:322 332
9 Springer-Verlag 1985
The production of callus capable of plant regeneration
from immature embryos of numerous Zea mays genotypes
D.R. Duncan, M.E. Williams, B.E. Zehr and J.M. Widholm
Department of Agronomy, University of Illinois, Urbana, IL 61801, USA
Abstract. In the summer of 1983, immature
embryos from 101 selfed inbred lines and
germplasm stocks of Zea mays L. were examined
for their ability to produce callus cultures capable
of plant regeneration (regenerable cultures) using
a medium with which some limited success had
previously been obtained. Forty-nine of the
genotypes (49%) produced callus which visually
appeared similar to callus previously cultured and
shown to be capable of plant regeneration. After
five months, 38 of these genotypes were alive in
culture and plants were subsequently regenerated
from 35 (92%) of them. No correlation was
observed between plant regeneration and callus
growth rate, the vivipary mutation (genes vpl, 2, 5,
7, 8 and 9), or published vigor ratings based on
K + uptake by roots. When F 1hybrid embryos were
cultured, 97% of the hybrids having at least one
regenerable parent also produced callus capable of
plant regeneration. No regenerable cultures were
obtained from any hybrid lacking a parent capable
of producing a regenerable callus culture.
In the summer of 1984, immature embryos from
218 additional inbred lines and germplasm stocks
were plated and examined for their ability to
produce regenerable callus cultures on media
containing altered micronutrient concentrations,
3,6-dichloro-o-anisic acid (dicamba), glucose, and
elevated levels of vitamin-free casamino acids and
thiamine. Of these genotypes 199 (91%) produced
callus that was regenerable in appearance. In the
1984 study, plant regeneration was noted in many
commercially important inbreds, including B73,
Mo17, B84, A632, A634, Ms71, Wl17, H993H95
and Cml05. Thus tissue-culture techniques are now
available to obtain callus cultures capable of plant
Abbreviations, trade names: 2,4-D=2,4-dichlorophenoxyacetic
acid; dicamba = 3,6-dichloro-o-anisic acid
Planta
regeneration from immature embryos of most
maize genotypes.
Key words: Embryo (tissue culture) Tissue culture
(plant regeneration) - Zea (plant regeneration).
Introduction
The regeneration of plants from tissue cultures of
Zea mays L. was first reported by Green and
Phillips (1975) utilizing immature embryos as the
tissue source. Using the same tissue system,
Springer et al. (1979) demonstrated that the plant
regeneration was by means of organogenesis.
However, Rice et al. (1978) and Lu et al. (1982)
found that plant regeneration could also occur by
somatic embryogenesis. Both types of regeneration
arise from hard, white or yellow callus that appears
distinctly different from the granular, gray-yellow
and translucent callus incapable of plant regener-
ation. Consequently, visual selection of regener-
ation-competent calli has become the method of
choice for detecting and maintaining plant
regeneration capability in Z. mays tissue cultures
(Springer et al. 1979; Tome et al. 1980; Armstrong
and Green 1982; Green 1982; Lu et al. 1982, 1983;
Rhodes et al. 1982; Sachs et al. 1982).
As published to date, there are only about a
dozen maize stocks that can be cultured and
maintained in a state capable of plant regeneration
(Hibberd 1984). These consist of a few inbred lines,
populations, F 1 hybrids and open-pollinated
hybrids (Green and Phillips 1975; Freeling et al.
1976; Harms et al. 1976; Green 1977; Torne et al.
1980; Rice 1982; Sachs et al. 1982; Earle 1983).
They do not include many commercially important
inbreds nor the vast majority of the maize
germplasm stocks. The culturing and regeneration
D.R. Duncan et al.: The production of callus capable of plant regeneration
of these other materials are essential if maize tissue
culture is to be effectively used in physiological and
genetic research.
The apparent lack of success with the majority
of the available maize material, as evident in the
limited number of publications, indicates: a) that
there is a definite genetic limitation to culturing
maize, b) that the media and-or the methods so far
used for culturing maize are inappropriate in most
circumstances, or c) that there has been an
inadequate survey of available material to ascertain
the culturability of maize. The work presented here
was carried out to study these three potential
problems of maize tissue culture.
The general approach to this research was
first to survey a large number of inbred lines,
germplasm stocks, populations and FI hybrids for
formation of regeneration-competent callus from
immature embryos formed using controlled
pollinations. The inbred lines and populations
could possibly indicate breeding stocks that have a
greater potential for culturing regenerable callus.
Similarly, the germplasm stocks of maize include
numerous mutants that, if cultured, might shed
some light on the genetic and physiological nature
of culture formation and plant regeneration. For
instance, precocious germination of seed on an ear
(vivipary) is induced by several genes (vpl, 2, 5, 7,
8, and 9; Neuffer et al. 1968) and perhaps the same
physiological system that operates to produce
vivipary is required to regenerate plants from tissue
culture. The production of cultures from germ-
plasm stocks possessing these and other genes may
elucidate the mechanism of plant regeneration
from maize tissue culture; consequently, such
germplasm stocks were included in the survey.
Over the past 50 years of breeding maize,
selections have produced modern maize lines with
less efficient uptake and assimilation of nitrate and
sulfate (Cacco et al. 1983). On the other hand,
Frick and Bauman (1978) found a positive
correlation between the heterosis expressed by
hybrids and the potassium uptake of those hybrids
and their parents. These observations indicate that
to culture a large number of maize lines, factors
such as the quantity and the type of nutrients made
available to the cultured tissue may need to be
altered to accommodate the variation in nutrient
demand seen at the whole-plant level. Consequent-
ly, modifications of media were examined to
determine what, if any, influence they might have
on tissue culture formation and plant regeneration
in maize; and a number of the lines used by Frick
and Bauman (1978) were included to examine any
possible correlations.
323
Materials and methods
Embryos used. Opaque, approx. 1.5-ram-long, immature
embryos were excised aseptically using methods similar to those
of Green and Phillips (1975) from irrigated plants of Zea mays
L. Embryos were harvested from cross-pollinated F1 hybrids,
self-pollinated inbred lines and germplasm stocks, and
randomly-mated RSSSC (Kauffmann and Dudley 1979) and
RSL X South African Composite (Alexander and Spencer 1982)
populations grown in the field during the summers of 1983 and
1984 in Urbana. Seed was obtained from Illinois Foundation
Seeds (tFS), Champaign, Ill., Holden's Foundation Seeds,
Williamsburg, Ia., from the Maize Genetics Cooperative,
Department of Agronomy, University of Illinois, Urbana, USA
or from other universities.
Summer 1983 survey. Fifty embryos collected from one to three
plants of each genotype were placed in presterilized, disposable,
plastic Petri dishes (100 mm diameter, 15 mm high) on approx.
25ml of "A" and "E" media (Table 1). All cultures were
incubated in the dark at 28~ and maintained with a 14-d
transfer cycle on "A" medium.
After five months in culture, plant regeneration was initiated
by transferring regeneration-competent callus onto either "E"
or "F" medium (Table 1), as suggested by Green (1982) and Lu
et al. (1982). These cultures were subsequently incubated at
28~ in continuous light (fluorescent lamps; F20TI2 cool-
white, Sylvania, Fall River, Mass., USA; approx. 80pmol
photons m-2s-1). Mature embryos with a visible coleoptile
developing on either of these media were transferred to "G"
medium (Table 1) in 150-ram-long, 25-ram-diameter test tubes
capped with Kaputs (Bellco Glass, Vineland, N.J., USA).
Plantlets which formed were transplanted into a 1:1 (v/v)
potting mixture of coarse Vermiculite and sieved peat moss in
4-cm-wide, 6.5-cm-deep conical compressed-peat-moss pots,
and were grown in a greenhouse until vigorous growth was
noted. Once roots appeared at the pot edge the plants, including
the pots, were transplanted to soil in the field or to a 3:3:1 (by
vol.) potting mixture of coarse Vermiculite, peat moss and field
soil in the greenhouse. Greenhouse conditions were a 14-h day
produced by 1000-W metal-halide iamps, 27~ day and 16~
night, and no humidity control. Plants were fertilized once a
week with a 20-20-20 (N-P-K) fertilizer (Robert D. Peters,
Allantown, Pa., USA) supplemented with the MgSO4 and
micronutrients of Hoagland's solution (Hoagland and Arnon
1950).
Summer 1984 survey. Thirty embryos collected from one to three
plants of each genotype were placed on "A", "B", "C" and "D"
media (Table 1). Calli were incubated as in 1983 but were
maintained on the medium which induced the most rapid callus
growth from the embryos.
Regeneration was initiated by transferring callus to "H"
medium (Table 1), instead of "E" and "F" media as was done
in 1983. Once plantlets developed, they were transferred to 25-
ram-diameter, 150-ram-long test tubes as in 1983 but with 17 ml
of Murashige-Skoog medium (1962) lacking growth regulators,
instead of medium "G". These changes in regeneration pro-
cedure had previously proven useful in decreasing the time
required to regenerate a plant from callus. When a substantial
root system and a shoot were formed (the shoot touching the
tube cap), the plantlet was transplanted to the greenhouse as in
1983.
Results and discussion
1983 survey. Calli initiated from immature embryos
on media "A" or "E" (Table 1) were visually
324 D . R . D u n c a n et al.: T h e p r o d u c t i o n o f callus capable o f p l a n t regeneration

Table 1. M e d i a used in 1983 a n d - o r 1984 e x p e r i m e n t s a

Ingredients Maintenance media Regeneration media

"A . . . . B. . . . C. . . . D. . . . E. . . . F. . . . G. . . . H"

N 6 macronutrients b + + + + + + + +
N 6 micronutrients b + + - _ + + + -
N 6 organic n u t r i e n t s b + + - - + + + -
B5 m i c r o n u t r i e n t s c - - + + - - -- +
2 , 4 - D i c h l o r o p h e n o x y a c e t i c acid 4.5 - 4.5 - 4.5 - - -
(2,4-D) d, g M
3,6-Dichloro-o-anisic acid - 15 - 15 . . . .
( D i c a m b a ) e, laM
L-proline d, m M 12 12 12 12 12 12 12 12
R T V i t a m i n s fig, ml 1-1 - - 1.0 1.0 - - - 1.0
E n z y m a t i c casein h y d r o l y s a t e d, g 1 I 0.1 0.1 - - 0.1 0.1 0.1 -
Vitamin-free c a s a m i n o acids h, g 1 1 _ _ 0.5 0.5 - - - 0.1
N a F e E D T A i, ml 1 1 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
KNO3, mM 28.0 28.0 28.0 28.0 28.0 28.0 28.0 28.0
T h i a m i n e - H C l d, g M - - 1. t 1.1 - - - 1.1
Sucrose, m M 60 60 60 60 360 180 60 60
G l u c o s e g, m M - - 56 56 - - - 56
Difco B a c t o - a g a r h, g 1-1 8.0 8.0 8.0 8.0 8.0 8.0 8.0 8.0
Activated charcoal k, g 1 1 . . . . . 5.0 5.0 -
p H before a u t o c l a v i n g 1 5.8 5.8 5.8 5.8 5.8 5.8 5.8 5.8

a Ingredients for N 6, B 5, N a F e E D T A a n d R T v i t a m i n s are listed in Table 2

b N 6 ingredients prepared a c c o r d i n g to C h u et al. (1975)
c B 5 ingredients prepared according to G a m b o r g et al. (1968)
d S i g m a C h e m i c a l Co., St. Louis, Mo., U S A
e Velsicol C h e m i c a l Co., Chicago, Itl., U S A
f R T V i t a m i n s p r e p a r e d according to H o r n et al. (1983)
g Filter-sterilized into m e d i u m after a u t o c l a v i n g
h Difco L a b o r a t o r i e s , Detroit, Mich., U S A
i N a F e E D T A (ethytenediaminetetraacetic acid) p r e p a r e d according to J a c o b s o n (1951)
k Fisher Scientific, St. Louis, Mo., U S A ; catalog No. C-170, c a r b o n , decolorizing (Norit)
I p H adjusted to 5.8 with K O H a n d H C l

Table 2. C o m p o s i t i o n o f n u t r i e n t m i x t u r e s used in Table 1

N 6 m a c r o n u t r i e n t s (raM) a N 6 m i c r o n u t r i e n t s (p,M) a N 6 organic n u t r i e n t s (ixM) a

( N H 4) 2SO 4 3.50 M n S O 4'H 2 0 19.73 Glycine b 26.64

K H 2PO 4 2.30 Z n S O 4"7H 2 0 5.22 Thiamine-HC1 b 2.96
M g S O 4"H 2 0 0.75 ' H 3BO 3 25.88 Nicotinic acid c 4.06
CaC12-2H 2 0 1.13 KI 4.82 Pyridoxine-HC1 b 2.44

B5 m i c r o n u t r i e n t s ( g M ) a RT vitamins (gM) d N a F e E D T A (pM) d

M n S O 4"H 2 0 59.17 Choline-HC1 b 0.70 N a 2 E D T A ' 2 H 2~ b, e 100.10

H 3BO 3 48.52 Riboflavin b 0.13 F e S O 4 7H 20 99.99
Z n S O 4"7H 2 0 6.96 D-Biotin 6 0.41
Na2MoO4'2H20 1.03 Folic acid b 0.11
C u S O 4"5H 2 0 0.10 Nicotinic acid c 1.62
CoC12"6H 2 0 0.11 Thiamine-HC1 b 0.30
KI 4.52 Ca p a n t o t h e n a t e c 0.21
Pyridoxine-HC1 b 0.97
Cyanocobalamin b 0.10 n M
p - A m i n o b e n z o i c acid b 0.36

a Final c o n c e n t r a t i o n s in m e d i u m
b S i g m a C h e m i c a l Co., St. Louis, Mo., U S A
c I C N N u t r i t i o n a l Biochemicats, Cleveland, O., U S A
d Stock c o n c e n t r a t i o n s ; stocks used as indicated in T a b l e 1
e E t h y l e n e d i a m i n e t e t r a a c e t i c acid; Sigma, catalog No. ED2SS
D.R. Duncan et al.: The production of callus capable of plant regeneration 325

Fig. 1. Regenerable (R) and non-regenerable (NR), 20-d-old

callus of Pa91 grown on medium " D ' . Bar= 1 cm

assessed, when 10-20d old, for regeneration-

competence in accordance with descriptions by
Rice etal. (1978), Green (1982), and Lu etal.
(1982). Regenerable calli were hard, irregularly
shaped, nodular, and white or yellow in color
(Fig. 1). Calli incapable of plant regeneration were
soft and appeared granular, translucent and gray-
yellow in color (Fig. 1). After five months in
culture, the initial assessments were verified by the
fact that whole plants were regenerated from 92%
of the genotypes that were scored as regenerable.
Genotypes which appeared to be regenerable but
grew too slowly to regenerate plants included L289,
B84 and Mo17. Regeneration appeared to be by
somatic embryogenesis as described by Rice et al.
(1978) and Lu etal. (1982), a scutellum and Fig. 2A, B. Embryos newly formed on medium "H" of 14-d-old
maize H99 callus; C=coleoptile, S=scutellum. A shows an
coleoptile at maturity being the primary evidence immature embryo while the embryo in B is commencing
of embryogenesis (Fig. 2). Embryogenesis was germination. Bar = 1 cm
particularly evident when "E" medium was used in
the regeneration procedure.
Of the 101 selfed inbreds or experimental and (Tables 3, 4, 7). In the case of callus growth rate,
mutant germplasm stocks tested, 49 (49%) both regenerable (H99, H60, W64A) and non-
produced regenerable calli (Tables 3, 4). Of the regenerable callus (A632, B14, germplasm stock
inbreds which were found to be regenerable only 987) grew very rapidly (Table 5). The viviparous
A188, A619, Wf9 and W64A had been shown mutants examined can be divided into two groups,
previously to be capable of plant regeneration vp2, 5, 7, 9 and vpl, 8. In the former group,
(Green and Phillips 1975). Even though other premature germination is repressed by abscisic acid
authors have reported success with Black Mexican (ABA) and seed color is white or pale yellow
(Sachs et al. 1982) and B37 and W22 (C.E. Green, whereas the latter group of mutants respond
Molecular Genetics, Minneapolis, Mn., USA, minimally to ABA and have normal seed color
personal communication), we were not successful (Fong et al. 1983). In some cases, lines from each
with these. group were capable of regeneration from culture
No clear correlations could be seen between (vp5, 7, 8; Table 3) and in other cases no
regeneration capability and callus growth rate regeneration was evident (vpl, 2, 9, Table 4).
(Table 5), vivipary (Tables 3, 4), or the vigor ratings In the case of K + uptake, Frick and Bauman
of Frick and Bauman (1978), which are based on (1978) rated B77 the highest and A632 the second
measurements of K + uptake by seedling roots lowest in their system of measuring K § uptake.
326 D.R. Duncan et al. The production of callus capable of plant regeneration

Table 3. Selfed inbreds and germplasm stocks of maize that Table 4. Selfed inbreds and germplasm stocks of maize from
produced regenerable callus cultures on "A" or "E" medium which no regenerable callus was obtained on "A" or '~ me-
in 1983 dium in 1983
Inbreds
Inbreds
Genotype Plants Genotypes Plants
formed formed A632 a B73 a Fr23 H98 a
Bt4a B77 Fr24 Hy2
A188 + H99 b + B14A CI-187-2 Fr25 R802A
A619 b a L289 - B37 a Frl6 Fr27rhm R805
A658 + L317 + B68 a Fr22 H84 a W23
B79 + M14 a
B84 - Mo17 b - Germplasm stocks
C103 a Oh7 +
CI540 a Os420 a Vp 1 FrB73/Teo 26 BSI-5878 SSI-5733
Fr20A a Pa91 + Vp 2 75-R091 BSI-5881 SSI-5739
H60 b + R806 + Vp 9 74-R215 BSI-5891 SSI-5745
H97 b + Wf9 + 474-2 W77-R035 BSI-5892 SSI-5782
W64A b § 987 RSC-129 BSI-5898 SSI-5784
Black Mex. Sweet BSI-5841 BSI-5902 SSI-5807
Silver Queen BSI-5859 BSI-5910 SSI-5820
Germplasm stocks B73/Teo 16 BSI-5868 SSI-5730 SSI-5826
Genotype Plants Genotype Plants a Lines used in study by Frick and Bauman (1978) of K +
formed formed uptake in maize

Bs10-676 a SSI-5744 a
Bs10-844 + SSI-5754 a Table 5. Growth a of embryo derived callus of several Zea rnays
Bsi-5867 a SSI-5755 + inbred lines
Bsl0-ca/SA Photo 3 + SSI-5773 +
(ETO./Ill.HEO2) - SSI-5780 + Callus diameter (mm)
S 2-Med. 2
Ill. Ear. Ear. Syn. + SSI-5825 +
Ill. High Oil + vp 5 a 2~4 4 7 7-10
Ill. High Prot. + vp 7 +
Ill. Low Prot. + vp 8 + A685 B37 A632
Ill. Rev. High Prot. + W77-R3019 + B73 B68 Black Mexican
Iojap + 38-11 + B77 H98 B14
RSC-98 + 121-9 + B84 Hy2 H60
RSC-520 a (1028 X 987)F2 + CI- 187-2 R802A H99
SA Photo 3 + (Mo17XH99)F2 + Mo 17 W64A
Oh7 987
a Culture lost before plants could be regenerated Oh43
b Lines used in study by Frick and Bauman (1978) of K + R805
uptake in maize W23

a Callus on the original explanted embryos grown for 30 d on

N e i t h e r o f t h e s e lines p r o d u c e d r e g e n e r a b l e c a l l u s
u n d e r t h e c o n d i t i o n s o f t h e 1983 s t u d y ( T a b l e 4).
O n t h e o t h e r h a n d , t h e y r a t e d A 6 1 9 as t h e s e c o n d
h i g h e s t a n d H 9 7 as t h e l o w e s t in t h e i r s y s t e m a n d
b o t h o f t h e s e lines p r o d u c e d r e g e n e r a b l e c a l l u s
( T a b l e 3). A s i m i l a r p a t t e r n c a n be seen in t h o s e
hybrids examined by Frick and Bauman that we
a l s o c u l t u r e d ( T a b l e 7). T h e h y b r i d B73 X H 9 9 w a s
ranked second highest while W64A X H99 was
ranked second lowest and both of these hybrids
p r o d u c e d r e g e n e r a b l e callus. O n t h e o t h e r h a n d ,
H98 X Mo17 was ranked highest while H98 X
A632 was ranked lowest and neither of these
h y b r i d s p r o d u c e d r e g e n e r a b l e callus.
Regenerable-callus production by two random-
mated maize populations was distinctly different
"A" medium was used to obtain callus diameter measure-
ments. Initial explants were approx. 1.5 mm long
( T a b l e 6). P o o r g r o w t h a n d o n l y s l i g h t l y re-
g e n e r a b l e c a l l u s w a s p r o d u c e d , in m o s t cases, f r o m
the RSSSC population (Kauffmann and Dudley
1979), a s t i f f - s t a l k p o p u l a t i o n s i m i l a r to t h o s e u s e d
to p r o d u c e t h e c o m m e r c i a l l y i m p o r t a n t i n b r e d s
B37, B73, B84, a n d f r o m w h i c h t h e S S I a n d R S C
lines w e r e d e r i v e d ( T a b l e s 3, 4). O n t h e o t h e r h a n d ,
the RSL X South African composite population
( A l e x a n d e r a n d S p e n c e r 1982), a m o d i f i e d L a n -
c a s t e r p o p u l a t i o n , p r o d u c e d t h r e e t i m e s as m u c h
r e g e n e r a b l e c a l l u s w h i c h , in g e n e r a l , g r e w r a p i d l y .
O f t h e i n b r e d lines p r o d u c i n g r e g e n e r a b l e callus,
3 3 % w e r e e a s i l y i d e n t i f i a b l e as b e i n g L a n c a s t e r -
D.R. Duncan et al.: The production of callus capable of plant regeneration 327

Table 6. Regenerable callus production of two random-mated Table 7. Regenerable callus formation in 1983 from Fl-hybrid
maize populations plated on medium "A" in 1983 immature embryos

Population No. of No. of embryos Regen- Genotype Cultured Genotype Cultured

ears erable embryos embryos
sampled Plated Forming callus forming forming
regen- formed embryogenic- embryogenic-
erable (%) appearing appearing
callus callus on callus on
medium "A" medium "A"
RSSSC a 165 4125 370 9.0 (%) (%)
RSL X South
African 114 2987 921 30.8 W64A X H99 c 40.0 H98 X Mo17 c 0.0
compositeb H99 X H60 c t00.0 W64A X Bt4 c 0.0
A632 X H99 c 10.0 B68 X H98 c 0.0
a Kauffmann and Dudley (1979) B73 X H99 c 3.0 B73 X Mo17 c 0.0
b Alexander and Spencer (1982) B68 X H99 c 6.5 B73 X H98 c 0.0
H99 X Mo17 c 33.3 B37 X Mo17 c 0.0
A188 X A619 a H98 X A632 c 0.0
population-derived (lines A619, C103, Fr20a, A188 X B84 73.5 (65.5) b
L289, L317, Mo17). On the other hand, only 10% A188 X Pa91 93.5 (89.5)b
A 1 8 8 X F r M o 1 7 71.5 (50.0)u
of the regenerable cultures were easily identifiable A188 X FrB73 15.6 (14.0) b
as being derived from a stiff-stalk population (lines A188 X RSC- 5.7 (0.0)b
B79, B84). For lines not producing regenerable 193
callus, 50% were from stiff-stalk populations (lines A188 X W64A 6.7
B73 X H97 c 3.0
A632, B14, BI4A, B37, B68, B73, B77, Fr27rhm,
H84 X H97 c 80.0
H84, R802A) and 25% were from Lancaster B37 X H97 c 3.0
populations (lines Fr22, Fr23, Fr24, Fr25, H98). Mo17 X H97 c 11.2
These data indicate that the capacity to form B37 X H60 c 66.7
regenerable callus cultures in maize has a heritable B73 X H60 c 3.0
Mo17 X W64A a
basis.
Examination of the pedigree of some of the a Percent embryogenesis could not be obtained since plants
regenerable inbred lines has shown that one or were regenerated from cultures initiated on medium "E"
more of the parental lines are also regenerable b Reciprocal cross
c Lines used in study by Frick and Bauman (1978) of K +
(where regeneration capacity has been tested): e.g.,
uptake in maize
R806, parental population Alexho (Freeling et al.
1976); A188, parental germplasm Silver King and
N.W. Dent (E. Green, personal communication); reciprocal crosses of A188 with other inbred lines
Pa91, parental lines Wf9, 38-11 and L-317 (Table (Table 7). With one exception, the frequency of
3); MolT, parental line C103 (Table 3); and W64A, regenerable callus formation by embryos from each
parental line Wf9 (Table 3). As with the population cross was similar to its reciprocal cross. The one
data, these pedigree observations (pedigree list; exception was the A188 X RSC-193 hybrid. In this
Henderson 1980) also indicate that, under the case, only 5.7% of the embryos plated produced
conditions described, plant regeneration in maize is regenerable callus, whereas the reciprocal cross
genetically determined. These observations also produced no regenerable callus. In general, this
have the potential utility of predicting the re- study of reciprocal crosses indicated that there were
generation capability of an untested line by probably no maternal effects regulating the
examining its parentage. regeneration process. We have also noted that calli
In studies of F 1 hybrids, regenerable callus was formed from these F~ hybrids generally grew faster
found in 97% of the cases where at least one of the than calli from inbreds.
parents exhibited regeneration (Table 7). The cross The dominant expression of plant regeneration
of W64A X B14 did not produce regenerable callus in a hybrid and the apparent expression of hybrid
(Table 7) even though the inbred W64A produced vigor in maize tissue cultures has been demon-
some regenerable callus. As with the population strated previously with one reciprocal cross by
and pedigree data, the Fl-hybrid results again Green (1977, 1981). The present data expand these
indicate a genetic basis for regenerable callus observations on the genetic control of regeneration
formation. to a larger number of hybrids, to populations, and
Of the F 1 hybrid combinations tested, five were to the parentage of inbreds in general.
328 D.R. Duncan et al.- The productionof calIus capable of plant regeneration
The data collected in 1983 demonstrate that a
wide variety of maize genotypes, when cultured on
"A" or "E" medium, produced calli capable of
plant regeneration. However, in many instances
these media did not support the production of
regenerable callus (Tables 4, 7) or even callus
growth (Table 5). Several cultures initiated from
embryos of Mo17 and B84 appeared to be
regenerable (Table 3), but their slow growth rate
prevented their maintenance and subsequent plant
regeneration. The growth of these cultures
indicated that possibly modifying the culture
medium was needed so as to stimulate the growth
of recalcitrant lines and to expand perhaps the list
of useful regeneration-competent maize genotypes.
1984 survey. The 1983 data indicated that mo-
difications of the culture medium might improve
the growth and plant regeneration capabilities of
maize inbred lines. The lack ofmolybdate, a known
cofactor of nitrate reductase, in the 1983 N6-based
media directed research efforts towards modifying
the nitrogen content of the media. Using a non-
regenerating suspension of Black Mexican sweet
corn developed by Chourey and Zurawski (1981),
preliminary experiments leaving out the reduced
nitrogen of medium "A" indicated that, at least for
this culture, the major sources of nitrogen available
to the cells were casein hydrolysate and proline.
Furthermore, substantial increases in growth could
be obtained by both increasing the casein hydro-
lysate concentration in the medium and by adding
molybdate to the medium (data not presented).
In the winter of 1983-1984 a survey was
conducted to examine embryo-derived callus
growth on medium modified in nitrogen content.
Preliminary results from that survey indicated that
increasing the available nitrogen in the medium
greatly enhanced growth (data not presented). The
use of vitamin-free casamino acids facilitated the
increase in reduced nitrogen in the medium while
maintaining a known level of vitamins. Except for
thiamine, which preliminary experiments indicated
was needed in greater quantities than previously
used, the vitamin requirements were not tested. The
addition of Bs micronutrients to the medium
supplied motybdate, copper and cobalt to the basic
N 6 medium.
In addition to examining the effects of nitrogen
sources and levels on the growth of embryo-derived
callus, various compounds with growth-regulating
activity were studied. These studies were prompted
by the report of Conger et al. (1983) indicating that
there were possibly better growth regulators
available for monocotyledons, such as maize, than
the often-used 2,4-dichlorophenoxyacetic acid
(2,4-D). Preliminary experiments indicated that, as
with orchardgrass (Conger etal. 1983), 3,6-
dichloro-o-anisic acid (dicamba), a chlorinated
benzoic-acid derivative, might be helpful in the
production and growth of regenerable maize callus.
The results of the winter survey produced media
"B", "C" and "D". These media contained di-
camba and-or elevated levels of reduced nitrogen
in the form of vitamin-free casamino acids, elevated
levels of thiamine, a complex mixture of vitamins
and glucose. In addition, molybdate, copper and
cobalt were added to the regular micronutrients of
the N 6 medium.
In the summer of 1984, another survey of maize
inbred lines was conducted, using both the old
("A') and new media ("B", "C", "D"; Table 1).
In this survey we examined some previously tested
lines but concentrated specifically on the commerci-
ally useful inbred lines listed by Zuber and Darrah
(1980), leaf-blight resistant lines (W. L. Pedersen,
personal communication), elite and experimental
lines of Illinois Foundation Seeds and Holden's
Foundation Seeds.
Since 92% of the callus that appeared to be
regenerable in the 1983 survey did indeed produce
plants after five months in culture, regenerability in
the 1984 survey was again based on visual
appearance of the calli. Of the 218 selfed inbred
lines and germplasm stocks tested, 199 (91%)
produced calli appearing capable of plant
regeneration (Table 8). Because of the numbers
involved and the fact that the calli produced were
to be used in other experiments not discussed here,
regeneration of each line producing what appeared
to be regenerable callus was not attempted.
Consequently, depending on the intended experi-
mental use, from 5 to 2000 plants were regenerated
from A619, A634, B73, B84, Bs10-676, F84-700,
F84-267, F84-275, F84-278, Fr22, Fr27rhm, H99,
LH51, LH123, Mo17, Ms71, Oh545, Pa91, Wl17,
W77-R3029A, 79-R4443, Down's Tester, Parker's
Flint, Wilber's Flint, and Tama Flint. These plants
were grown in either a greenhouse or a Florida
nursery, and yielded normal-looking plants in
terms of height, tassel and ear appearance, and
pollen shed. The F84 series produced ears in place
of tassels in 2% of the plants; this had not been
seen under normal growing conditions with plants
grown from seed.
In 1983, 49 % of all lines tested on medium "A"
formed regenerable callus; in 1984 the yield was
54% on medium "A" (Tables 9, 10). While the
results obtained in the two years with medium "A"
are very similar, they are lower than those obtained
with the modified media ("B', "C", "D") tested
D.R. Duncan et al.: The production of callus capable of plant regeneration 329

Table 8. Selfed inbreds and germplasm stocks of maize that Table 9. A summary of maize embryos producing regenerable
produced regenerable callus culture in the 1984 survey a, b callus in the 1984 survey

Inbreds No. of embryos producing regenerable callus/

No. of embryos plated
A545trf F84-252-3 Fr29 R168
A554 F84-267 Fr31 R802w "A . . . . B. . . . C. . . . D"
A619Ht2 F84-275 Fr35 Roh43HtA
A632 c F84-278 Fr52 Rw64AHtA Generalsurvey 710/3202 933/3131 1 112/2960 1462/3121
A632wx F84-440 Frl40 Tama Flint Holden's 19/345 55/345 95/360 84/315
A634 F84-453 Fr303 Tx601 (LH lines)
A635 F84-459 Fr619 Va26Ht
A637 F84-700 FrVa26 W23 AC Stock 2/169 1/119 2/105 1/75
A639 F84-729 H49 W77-R3021 Sweet Corn 552/1545 465/1430 749/1623 882/1626
A654 F84-762 H93 W77-R3025 (sh2 mutants,
A664 F84-765 H98 c W77-R3027 F84 lines)
A665 F84-767 H993H95 W77-R3029A Miscellaneous 48/305 78/385 83/380 81/395
B37 c F84-709-1 Hy2 c W77-R3048 (Pro 1,
B37Ht2 F84-803 Ky228 W77-R3288 ADH null)
B64 F84-812 LH51 Wl17
B73 c F84-820 LH 123 W 117Ht Mu stocks 20/209 71/235 136/236 122/201
B84 c F84-1147 LHI45 W153R [FS Lines 1 4 9 / 1 2 4 0 223/1174 359/1308 386/1280
Bs10-676 F84-1224-1 LHI50 Wilber's Flint (FR lines)
C103 c F84-1231 Mo17 c 50783
Cl03Dtrf F84-1242-I Ms71 52746 Total 1500/70t5 1826/6819 2536/6972 3018/7013
CH591-36 F84-1327 Mt42 75-R333 Regenerable 21.38 26.79 36.37 43.03
CM105 F84-1399 Mang. Tester 78-5048 (%)
CM109 Fr2A N28 78-5159
CM174 Fr2B N7A 78-R375
Co109 Fr4A ND203 79-1130
Co220 Fr4CA N28Htrhm 79-A1282 Table 10. A summary of maize lines showing regenerable callus
Down's Tester Frl4A Oh07B 79-R0090 in the 1984 survey
F84-153-1 Fr20A c Oh545 79-R0098
F84-168-1 Fr22 c P887p 79-R4326 No. of lines showing regenerable callus/
F84-176 Fr23 ~ Pa762 79-R4443 No. of lines plated
F84-183 Fr27rhm c Parker's Flint 83-40,191
F84-214 "A . . . . B. . . . C. . . . D"

Germplasm Stocks General survey 68/126 89/129 97/121 109/125

Holden's 1/7 3/7 4/7 4/7
ADH null 02846487 02846506 SSI- 17 c (LH lines)
bx/bx 02846488 02846507 SSI-56 c
AC Stock 1/1 1/1 1/1 1/1
Bx/Bx 02846490 02846509 SSI-92 c
02846476 02846492 02846510 SSI-99 c Sweet Corn 22/29 25/29 30/30 30/30
02846478 02846493 02846511 Stock 6 (sh2 mutants,
02846479 02846495 02846512 wxBfl F84 lines)
02846480 02846498 pro 1121 w2 Flint Corn 2/3 0/3 3/3 3/3
02846482 02846501 pro 1058 842347
02846483 02846502 SSI-2 c 842350 Miscellaneous 5/6 6/6 6/6 6/6
02846486 02846503 SSI-5 c 842351 (Pro 1,
ADH null)
a Does not include lines that were successfully cultured in 1983 Mu stocks 4/5 5/5 5/5 5/5
b Lines unsuccessfully cultured: A619Ht3, A656, A659, B68 c, IFS Lines 10/26 14/26 19/27 24/26
B68Ht, B37trf, CI66, Frl6, FrK55Ht, H95, K55, LH38, (FR lines)
LH74, LHI19, Oh509A, 02846475, 02846485, 02846491,
02846497, 02846499, 02846505, 02846508, Rw153RHtA, Total 113/203 143/206 165/200 183/203
SSI-79, T220, W77-R3047
c Lines unsuccessfully cultured in 1983 and retested in I984 Regenerable 55.67 69.42 82.50 90.15
(%)

in 1984. When 23 lines which produced no percent of lines producing regenerable callus,
regenerable callus in 1983 were again tested in 1984, however, were less than values obtained in the
approx. 83% of the lines produced regenerable general survey (Tables 9, 10, 11). The lines chosen
callus (lines used are indicated in Table 8). In these for retesting were derived from populations similar
cases, the quantity of callus produced and the to the RSSSC stiff-stalk population (Table 6) and
330
Table 11. Number of embryos and maize lines producing regenerable callus from 23 inbred lines grown in both 1983 and 1984
Media
No. of embryos producing regenerable
callus/total embryos plated
Yield of regenerable callus (%)
No. of regenerable lines/total lines plated
Yield of lines (%)
the lower values of regenerable callus production
are consistent with the observed poor culturability
of lines selected from the stiff-stalk population.
Some success (44%) was obtained with medium
"A" in 1984 as compared with 0% in 1983. This
44% value, however, was still lower than the 83%
obtained with the modified media. The reason for
the higher values in 1984 on medium "A" might be
that the 1984 growing season was more favorable
in comparison with the dry conditions during
anthesis in 1983.
The extreme alterations in the composition of
the culture media ("D" versus "A") had the effect
of nearly doubling the number of maize lines
producing regenerable callus (Table 10) and
doubling the quantity of regenerable callus pro-
duced (Table 9). As seen in the 1983 survey, seldom
did every embryo plated produce regenerable
callus. The 1984 production varied from 1.0% (e.g.
B73) to 100% (e.g. H99) of the embryos plated.
The number of genotypes which formed
regenerable cultures was increased with the use of
media "B" or "D", where dicamba was substituted
for the 2,4-D of media "A" and "C", respectively
(Table 10). With the addition of dicamba to these
media, there was also a decrease in the amount of
shoot formation and callus browning which was
usually produced by 20- to 30-d-old callus grown
on media containing 2,4-D. Dicamba also
increased the time required for plantlet formation
on regeneration media by approx. 7 d. In addition,
several genotypes behaved differently in the
presence of the two herbicides; an extreme example
of this was A554. In this case, the harvested
immature embryos germinated directly on the
media containing 2,4-D, while on media containing
dicamba, embryo germination was suppressed and
typical regenerable callus was formed. These results
indicate that either dicamba is a more powerful
growth regulator than 2,4-D at the concentrations
used or it is not metabolized as rapidly, thus
remains active longer. The literature supports the
D.R. Duncan et al.: The production of callus capable of plant regeneration
1983 1984
"A . . . . B. . . . C. . . . D"
10/1105 58/895 124/871 143/813 222/906
0.91 6.86 14.24 17.59 24.50
0/20 10/23 13/23 14/23 19/23
0.0 43.88 56.92 60.87 82.81
latter conclusion. Crafts (1964) reported that the
chlorobenzoic acids such as dicamba were not
altered by plants, whereas the chlorophenoxy acids
such as 2,4-D were readily metabolized.
Extensive modifications of the basic N 6 medium
with elevated levels of thiamine, reduced nitrogen,
and the addition of glucose and a wide assortment
of vitamins, as in media "C" and "D" (Table 1),
increased callus production and increased the
number of regenerable lines cultured more than the
dicamba substitution alone (Tables 9, 10). These
media changes (media "C", "D"; Table 1) also
stimulated the growth of many lines, e.g, Mo17,
B84 and C103, that would not grow on media "A"
and "B" (Table 5). At least in the case of these lines,
which appeared regenerable in 1983 but would not
grow, the formation of a usable regenerable culture
would seem to be solely associated with supplying
the nutrition needed for growth and not in the
induction of some "regeneration activity" that
previously was not present.
Groups of genotypes also responded differently
on the various media tested. For example, sweet
corn (F84 lines) in general produced large
quantities of regenerable callus on any of the media
(Tables 9, 10). On the other hand, IFS lines and
Holden's lines (Tables 9, 10) showed less re-
generation capability and media-dependent dif-
ferences in both callus production and regenera-
bility. The sweet corn lines (sh2 mutants) tested are
derived from Flint corn, and of the limited material
tested, Flint corn lines produce regenerable callus
abundantly (Table 10), indicating again that
regeneration-competence is in some manner
heritable. It is also possible that the sh2 mutation
may be beneficial to plant regeneration but isogenic
stocks with and without sh2 have not yet been
tested.
A similar pattern is seen with the IFS and
Holden's material. Many of these lines are derived
for commercial use from the commercially
important stiff-stalk background (Fr27rhm, Fr31,
D.R. Duncan et al.: The production of callus capable of plant regeneration
Frl40) or from the more difficult-to-culture
inbreds, such as Mo17 (Fr20A, Fr22, Fr23, Fr24,
Fr303). Both groups of lines produced fewer
regenerable cultures and smaller quantities of
regenerable callus than other groups of maize
tested (Tables 9, 10). This corresponds to the low
productivity of stiff-stalk-derived lines in 1983 as
compared with Lancaster-population-derived lines.
As in 1983, the 1984 data indicate that the parental
inbreds or parental population of these maize lines
greatly influence the production of regenerable
callus cultures. In the examples cited, all the lines
did grow and in most cases produce regenerable
callus on medium "D".
Conclusion
As stated above, the 1983 survey indicated that
there are genetic differences in the potential of
cultured maize lines to grow and regenerate plants.
The data from the 1984 survey indicate that the
expression of these genetic differences can either be
circumvented by or is related to the available
nutrients and growth regulators in the culture
medium. This was particularly evident when the
levels of reduced nitrogen and thiamine were
elevated and glucose, a wide variety of vitamins and
dicamba were added to a basic N 6 medium
(medium "D"; Table 1). Furthermore, these
studies indicate that when the source plant of a
tissue culture is grown under proper conditions
even poorly regenerating genotypes can be cultured
if a large number of embryos are harvested and the
calli capable of regeneration are visually selected
and maintained.
Thanks are extended to Tijana Knezevic, Miao Shu-hua,
Shanker Kothari, Damares Monte and Linda Wagner for their
technical assistance, and to R.J. Lambert, D.E. Alexander, G.F.
Sprague, G.B. Fletcher, J.W. Dudley, E.B. Patterson, and W.L.
Pedersen for valuable discussion, seed supplies and field
assistance. This research was supported by funds from the
Illinois Agricultural Experiment Station, Urbana and Illinois
Foundation Seeds, Inc., Savoy, Ill., USA.
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