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Michigan State
University

 

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This is to certify that the

thesis entitled
Clonal Propagation of the Common Bean,

Phaseolus vulgaris L., from Shoot-Tips

 

in Tissue Culture

presented by

Mary M. Saam

has been accepted towards fulfillment
of the requirements for

M. S. degree in Crop and Soil Sciences

 

 

 

Date 17 October, 1983

 

0-7539 MS U is an Affirmative Action/Equal Opportunity Institution

 

 

 

 

 

)VifSI_J BEIURNING MATERIALS:
Place in book drop to
LIBRARJES remove this checkout from
Jll-IC-ll-L your record. FINES will

be charged if book is
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stamped below.

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CLONAL PROPAGATION OF THE COMMON BEAN, PHASEOLUS
VULGARIS L., FROM SHOOT-TIPS IN TISSUE CULTURE

3)!
Mary Margaret Anne Saam

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Crop and Soil Sciences

1983

ABSTRACT

CLONAL PROPAGATION OF THE COMMON BEAN, PHASEOLUS
VULGARIS L., FROM SHOOT-TIPS IN TISSUE CUEIURE

BY

Mary Margaret Anne Saam

To overcome the problem of obtaining a large number of copies
of F1 genotypes from hand pollinations, an in vitro propagation method

for the common bean (Phaseolus vulgaris L.) was investigated. Clones

 

were derived by culturing apical shoot-tips 1.0 to 1.5 cm in length
on a modified Murashige and Skoog (1962) medium. The effects of two
cytokinins (kinetin, benzyladenine) and two auxins (indoleacetic acid,
naphthaleneacetic acid) alone or in combination were examined with
regard to shoot multiplication, root and callus formation. Responses
were evaluated and tissue transfers to fresh media occurred every 3
to 4 weeks. Combinations of benzyladenine (1.0 and 3.0 mg/l) and
naphthaleneacetic acid (0.1 mg/l) were optimal for shoot multiplica-
tion and could sustain long term (3 to 4 months) jg_yjtrg bean shoot
proliferation. Basal media with or without kinetin (0.5 mg/l) and
either indoleacetic acid (0.3 mg/l) or naphthaleneacetic acid (0.6
mg/l) was suitable for rooting cloned bean plantlets. At higher con-
centrations of both benzyladenine and kinetin (above 10 mg/l) shoot
production as well as internode elongation decreased sharply and

rosette-like cultures with many multiple buds formed. Most of these

Mary Margaret Anne Saam

cultures could not survive more than one transfer to fresh media.
Plantlets rooted on low concentrations of cytokinin and auxin

had a high survival rate and were acclimated to soil conditions with
a sterile potting procedure. All tissue culture derived plants were
grown to maturity in the greenhouse or field and produced fertile

flowers, pods and seeds.

TABLE OF CONTENTS

Page
LIST OF TABLES . . . . . . . . . . . . . . . . iii
LIST OF FIGURES. . . . . . . . . . . . . . . . vi

INTRODUCTION. . . . . . . . . . . . . . . . . l
REVIEW OF LITERATURE . . . . . . . . . . . . . . 3
MATERIALS AND METHODS. . . . . . . . . . . . . . ll

Plant Materials and Whole Plant Cultural Conditions. . . ll
In Vitro Culture Methods. . . . . . . . . . . . ll

E—plant Evaluation System . . . . . . l3
Acclimation of Plantlets to Soil Environment. . . . . l6
Field Experiments . . . . . . . . l7

Characteristics of Tissue CULture Plants . . . . . . 19
Statistical Analysis . . . . . . . . . . . . . 20

RESULTS . . . . . . . . . . . . . . . . . . 29

Initial Studies. . . . . . . . . . . 29
Cytokinin Concentration Gradients. . . . . . . . . 29
Cytokinin - Auxin Combinations. . . . . . . . . . 42
Final Media Selections . . . . . . . . . . . . 72
Light . . . . . . . . . . . . . . . . 82

In Vitro Rooting . . . . . . . . . . . 82
AEclimat tion to Soil Environment . . . . . . . . . 86
Field Experiments . . . . . . . . . . . . 86

DISCUSSION . . . . . . . . . . . . . . . . . 94
BIBLIOGRAPHY. . . . . . . . . . . . . . . . . 99

ii

TABLE

6a

6b

l0

ll

LIST OF TABLES

Morphogenetic responses of some legumes in tissue
culture. . . . . . . . . . . . .

Cultivars of common bean used in tissue culture
experimnts O O O O O O O O I I O I O

The major organic substrates and their concentrations
used in shoot multiplication and rooting media.

Conditions for soil acclimation.
Effect of BAP on cultured bean shoot-tips

Effect of low concentrations of kinetin on bean
shoot-tips. . . . . . . . . .

Effect of high concentrations of kinetin on bean
shoot—tips.

Optimal media for shoot propagation selected from
each two-way grid (BAP x NAA, BAP x 1AA, kinetin x
NAA and kinetin x IAA) and the growth responses of
bean apical buds after 20 days . . . .

Analysis of variance for the in vitro production of
shoots, roots and callus and tota ls hoot length of
bean apical buds cultured on kinetin and NAA .

Analysis of variance for the in vitro production of
shoots roots and callus and total shoot length of
bean apical buds cultured on BAP and IAA. .

Analysis of variance for the in vitro production of
shoots, roots, and callus and— tot tal length of bean
apical buds cultured on BAP and NAA . .

Analysis of variance for the in vitro production of
shoots, roots and callus and total l ength of bean
apical buds cultured on kinetin and IAA. . . .

Page

12

IS
18
30

3]

32

45

46

47

43

49

TABLE
12

13

14

15

16

17

18

19a

19b

20

21

22
23

24

Means of shoot production, shoot length, callus
formation and percent rooting of bean apical buds
cultured on BAP and NAA. . . . . . . .

Means of shoot production, shoot length, callus
formation and percent rooting of bean apical buds
cultured on BAP and IAA. . . . .

Means of shoot production, shoot length, callus
formation and percent rooting of bean apical buds
cultured on kinetin and NAA . . . . . .

Means of shoot production, shoot length, callus
formation and percent rooting of bean apical buds
cultured on kinetin and IAA . . .

Percent root formation of two common bean cultivars
grown on different combinations of kinetin and NAA

Percent root formation of two common bean cultivars
grown on different combinations of kinetin and IAA.

Primary trifoliate leaf development of two common bean
cultivars grown on different combinations of kinetin
and IAA . . . . . . . . . . . . .

Effect of media (BN3, BIZ, KN3 and K13) and the number
of subcultures on shoot-tip propagation of Fleetwood .

Effect of media (BN3, BIZ, KN3 and K13) and the number
of subcultures on shoot-tip propagation of UIlll

Effect of media (BN3, BIZ, KN3, K13) and the number of
subcultures on jn_vitro root formation of Fleetwood
and UIlll shoot cultures

formation after 2 weeks in culture .

 

Effect of kinetin, NAA and IAA on in vitro bean root

Male fertility of tissue culture derived plants.

Percent germination of seed obtained from ig_vitro
propagated bean plants . .

Analysis of variance for flower and pod formation of

bean plants grown from tissue culture derived seed
(P1 seed) . . . . . . . . . . . . . .

iv

Page

50

51

52

53
68

69

7O
73

74

75

85
90

91

92

TABLE Page

25 Means of flower and pod fbrmation of plants grown
from tissue culture derived seed (P1 seed) . . . . 93

LIST OF FIGURES

FIGURE Page
l Terminology used for seed derived from tissue culture
propagated plants. P = seed from plants cloned jg_ 14
vitro. . . . . . . . . . . . . .

2 Stages of the shoot multiplication system. . . . . 21
3 Stages of soil acclimation procedure . . . . . . 25

4 Effects of cytokinin concentration of shoot production
of UIlll apical buds. . . . . . . . . . . . 33

5 Effects of cytokinin concentrations of basal callus
formation from UIlll apical buds. . . . . . . . 34

6 Effect of kinetin concentrations on basal callus
formation from Fleetwood apical buds . . . . . . 35

7 Multiple buds which differentiated at a single axial
of a Tuscola shoot culture grown 50.0 mg/l BAP . . . 36

8 Morphologic responses of in vitro cultured UIlll and
Fleetwood apical buds grown on 75.0, 100.0 and 150.0
mg/l kinetin . . . . . . . . . . . . . . 39

9 A. Bean shoot-tips cultured on 0.3 mg/l BAP
B. Bean shoot-tips cultured on 30.0 mg/l BAP . . . 43

10 Effects of kinetin, BAP and NAA on shoot formation,
shoot length and callus production of Fleetwood apical
buds . . . . . . . . . . . . . . . . . 54

11 Effects of kinetin, BAP and NAA on shoot fbrmation,
shoot length and callus production of UIlll apical
buds . . . . . 156

12 Effects of kinetin, BAP and IAA on shoot formation,

shoot length and callus production of Fleetwood apical
buds . . . . . . . . . . . . . . . . . 58

vi

FIGURE
13

14

15

16

17

18

19

20

21

22

23

24

Effects of kinetin, BAP and IAA on shoot formation,
shoot length and callus production of UIlll apical
buds . . . . . . . . . . . . . . . .

Adventitious buds arising from callus of Fleetwood
ramets cultured on 3.0 mg/l BAP and 0.1 mg/l NAA

A. Bean plantlets cultured on BIZ and BN3
B. Bean plantlet cultured on BN3

Primary trifoliate leaf development of bean shoot-tips
cultured on 3.0 mg/l kinetin and 0.05 mg/l IAA . . .

Effects of media (BN3, BIZ, KN3, K13) and number of
subcultures on shoot production of Fleetwood tissue
cultures. . . .

Effect of media (BN3, BIZ, KN3, K13) and number of
subcultures on shoot production of UIlll tissue cul-
tures. . . . . . . . . . . . . .

Effects of media (BN3, BIZ, KN3, K13) and number of
subcultures on leaf production of Fleetwood tissue
cultures. . . . . . . . . . .

Effect of media (BN3, BIZ, KN3, K13) and number of
subcultures on leaf production of UIlll tissue cul-
tures. . . . . . . . . . . . .

Effect of media (BN3, BIZ, KN3, K13) and number of
subcultures on leaf area of Fleetwood tissue cultures.

Effect of media (BN3, BIZ, KN3, K13) and number of
subcultures on leaf area of UIlll tissue cultures .

Fleetwood bean shoots grown on BN3, BIZ, KN3 and K13
after 100 days in culture and 5 subcultures onto fresh
media. . . . . . . .

A. Fully acclimated tissue culture derived Tuscola
bean plant nearing maturity

B. Fully acclimated tissue culture derived Swan
Valley and Tuscola bean plants .

vii

Page

60

63

64

71

76

77

78

79

80

Bl

83

87

INTRODUCTION

Grain legumes are an important food crop because they supply
a good amount of dietary protein to consuming populations in many
countries of the world. Plants of common bean (Phaseolus vulgaris
L.) are highly self-pollinating and alleles of most loci are in the
homozygous state. Moreover, in nature self-pollination in beans is
insured by a unique flower morphology. Artificial cross-pollinations
are tedious and difficult to make. The lack of suitable genetic
or cytoplasmic male-sterility system in P;_vulgaris has forced
breeders to effect cross-pollinations by hand. The problem of obtain-
ing a large number of hand-pollinated crosses, is the poor seed set
in pods where cross-pollinations are successful. The lack of effi-
ciency in obtaining cross pollinated seed has not allowed breeders to
use recurrent selection effectively in bean improvement programs
(Kenworthy and Brim, 1979).

One of the alternatives to making many hand cross-pollinations
to obtain sufficient hybrid seed for recurrent selection or genetic
studies in beans is in vitro propagation. Such a system would produce
large populations of genetically uniform heterozygous types. Clones
that could be produced rapidly would provide sufficient numbers of
plants for evaluations and would reduce the time needed to complete a

cycle of selection (Kenworthy and Brim, 1979). In addition, the

ability to maintain plants jg yjtrg_would aid in metabolic studies
concerning certain secondary plant compounds. It would also provide
important information concerning growth and development in legume
cell and tissue cultures.

In 11:39 clonal propagation of many agronomic crop plants has
not been used because conventional seed propagation techniques to
insure genetic purity are more efficient, inexpensive, and practical.
In crops such as the cereals, forage legumes and grain legumes, large
acreages are usually grown. Bulk populations that are homozygous
and comprised of homogeneous or heterogeneous individuals are main-
tained by self-pollination and conventional seed propagation. How-
ever, in the breeding of certain crop plants, such as sugarbeet (Beta
vulgaris L.) and a number of pasture grasses, cloning is commonly
practiced to sustain unique materials (Conger, 1970; and Saunders,
1982). In many self-pollinating crops, where male sterility is not
available, the production of hybrids is difficult and expensive.

With the use of tissue culture, unique hybrids could be propagated
by using a rapid jg_yjtrg_clonal propagation scheme.

The purpose of this study was to assess the feasibility of
multiplying common bean genotypes via an jg_yjtrg_clonal propagation
scheme. Specific objectives were 1) to develop a suitable shoot
propagation technique from lateral and apical buds, 2) make recom-
mendations for multiplication of explants and define a rooting media;
for plantlet recovery and acclimation to greenhouse conditions and,
3) evaluate the use of the procedure for whole plant production from

tissue culture.

REVIEW OF LITERATURE

Murashige (1974) suggested that the propagation of valuable
plant materials could be accomplished by tissue culture techniques
which might be adapted for use in crop improvement programs. '12
XIEEQ propagation has been used commercially to asexually propagate
a large number of horticultural species where seed propagation is
either difficult or results in undesirable genetic segregation and
non-uniform plant populations (Evans and Sharp, 1982; Oglesby, 1978).
The goal of asexual propagation is to produce uniform plants of
unique genotypes (Murashige, 1974). When cultivars can easily be
propagated through conventional means, the application of tissue
culture can significantly enhance the rate of multiplication
(Murashige, 1974). It has been shown in pineapple (Zepeda and Sagawa,
1981) that conventional vegetative propagation produced only 5
cuttings per crown per year, whereas when tissue culture was used
for the same period of time 500 plantlets were obtained from a
single crown.

Murashige (1974) outlined a procedure for plant propagation
via tissue culture which had three stages. These were: 1) establish-
ment of an aseptic culture, 2) multiplication of propagula and 3)
preparation for plantlet reestablishment in soil (Murashige, 1974).

The first stage involved the selection of a suitable explant for

propagation and a sterilization procedure. Shoot-tips or apical
meristems were the most commonly used initial explants for most
species. In the rapid multiplication of propagules, the increase
was achieved via axillary shoot multiplication or by adventitious
organogenesis. When adventitious organogenesis was used,Murashige
(1974) stated there was a chance of producing genetically aberrant
plants, whereas with axillary shoot multiplication, the incidence
of genetic abnormalities was low. A defined nutrient medium was used
for both of these stages (Murashige, 1974). The final stage of
Murashige's (1974) procedure for in gitrg_propagation was the accli-
mation of tissue culture derived plants to the soil environment.
The purpose of this stage was to root, harden and convert the
plantlets from the heterotrophic to the autotrophic state.

Meristem culture is an important area of’jg_xjtrg_propagation.
This technique has been used for the production of disease-free plants,
in long term preservation of germplasm and for micropropagation of
unique lines (Kartha, 1982). Meristem culture is similar to shoot-
tip propagation; however, much smaller initial explants are
used. Depending on the species, a typical meristem-tip used in the
culture of seed propagated crops measures between 0.3 to 0.5 mm in
length (Kartha, 1981), whereas explants used in shoot-tip propagation
are normally 1.0 to 10.0 mm or longer (Hollings, 1965; Stone, 1978).
In 315:9 attempts to propagate, preserve and eliminate diseases from

two legumes: red cloVer (Trifolium repens L.) and pea (Pisum

 

sativum L.) have been successful (Phillips and Collins, 1970;

Bhojwani, 1981; Barnett et al., 1975; Kartha, 1974 and 1979).

One of the barriers limiting the manipulation of grain legume
species via tissue culture is the lack of information regarding their
jg_vitrg growth requirements. Murashige (1974) divided the nutrient
requirements of the media used in plant tissue culture into three
categories; inorganic salts, organic substances and natural complexes.
The inorganic salt requirements for jg_vjtrg shoot-tip propagation
have been consistent fbr most species studied and the Murashige and
Skoog (MS) salt formulation (1962) has been the most frequently used.
In a number of studies by Mok, et al., (1978a, b; 1979, and 1982)
concerning the effects of growth regulators on bean callus prolifera-
tion and the culture of interspecific Phaseolus hybrid embryos, the
MS (1962) basic salt formulation was used. Kartha (1981) also used
MS (1962) salts in his study of bean plantlet regeneration from
shoot meristems. The use of other mineral salt formulations has
been reported. Crocomo (1975) used the salt formulation of Veliky
et al., (1970) in a".ifl.!i££2 study of bean root, plantlet and
callus growth.

The organic substances which are important to the nutrient
media are carbohydrates, vitamins and growth regulators (Murashige,
1974). Sucrose is the most common carbohydrate source, although
glucose and fructose have also been used successfully (Murashige,
1974). The concentration of sucrose used in the culture of legumes
ranged between 20 (Phillips and Collins, 1979a, b; 1980) and 30
(Kartha, 1981; Beach and Smith, 1979) grams per liter.

Vitamin requirements vary from species to species, of those
most frequently used are: 1) inositol, 2) nicotinic acid, and 3)
pyridoxine (Murashige, 1974). Researchers working with soybean

[Glycine max L. Merr. (Cheng et al., 1980; Saka et al., 1980)], cow-

 

pea [Vigna unguiculata L. Walp. (Kartha, 1981)], peanut [Arachis

 

hypogaea L. (Kartha, 1981)], chickpea [Cicer arietinum L. (Kartha,

 

1981)], bean [Phaseolus vulgaris L. (Kartha, 1981)], alfalfa [Medicago

 

satixa_L. (Cheyne and Dale, 1980)], red and crimson clover [Trifolium
rgpgns L. (Beach and Smith, 1970; Cheyne and Dale, 1980)] all have
successfully used the vitamins in Gamborg's BS (1968) media (100
mg/l inositol, 1 mg/l pyridoxine, 1 mg/l nicotinic acid and 10 mg/l
thiamine).

A number of different amino acids and amides have been found
to be advantageous in some tissue cultures (Murashige, 1974).
Gamborg, et al., (1968) found that the addition of between 3 to 8
mM glutamine was beneficial for the growth of soybean suspension
cultures. This compound has also been useful in the culture of
immature embryos from interspecific hybrids between Phaseolus vulgaris

and E; lunatus and P; acutifolius (Mok, et al., 1978).

 

The most significant substances used in shoot propagation
media are the growth regulators or plant hormones (Murashige, 1974).
Levels of the auxins and cytokinins (two major types of hormones)
vary for each species being studied. Previously, researchers have
demonstrated that in shoot culture media, a high concentration of

cytokinin stimulates multiple bud formation (Murashige, 1974) but a

high concentration of auxin favors root initiation. Another hormone,
gibberellic acid, has also been found to be useful in stimulating
growth in some plant tissue cultures (Gautheret, 1969; Murashige,
1961 and 1964; Skene, 1969). The growth regulators used in the
tissue culture of several legumes are listed in Table l.

The practical use of tissue culture of legumes involves the
acclimation of in_vitrg plantlets to a greenhouse or field environment.
In several investigations concerning hybrid Phaseolus embryo rescue,
difficulty in adapting the hybrids to soil and greenhouse environ-
ments was encountered (Mok, et al., 1978a; Honma, 1955 and 1956).
Murashige (1974) has stated that research concerning the rooting,
transplanting and hardening of tissue cultured plants has been
neglected by many investigators, and more information about this stage
of ifl.!i£[9 propagation is clearly needed.

There have been very few attempts to actually propagate grain
legumes via tissue culture (Kartha et al., 1981) evaluated the shoot
apical meristem regeneration potential of bean. He found that at l
to 10 uM BAP bean meristems proliferated and produced multiple buds.
Maximum bud formation occurred at 10 uM BAP and as the concentration
was reduced to 5 or 1 pM, the number of buds differentiating also
decreased. He obtained whole plants when the meristems were cultured
on a medium supplemented with 1 uM NAA alone, and with 5 uM BAP and
10 uM NAA, multiple buds, callus and root formation was stimulated.
However, these multiple buds and shoots did not develop into whole

plants. When the multiple buds were subcultured to media containing

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10

1.0 pM BAP, 0.1 uM NAA and 0.1 pM gibberelic acid (6A3), shoots
elongated without differentiating roots. At this point Kartha,

et al., (1981) successfully rooted the elongated shoots on a half-
strength MS (1965) medium supplemented with 1 pM IAA, the plantlets

were then transplanted in soil and grown to maturity.

MATERIALS AND METHODS

Plant Materials and Whole Plant Cultural Conditions
Cultivars of common bean (Phaseolus vulgaris L., 2n = 22)

were used in all tissue culture experiments (Table 2). Seeds of

three navy bean cultivars; 'Fleetwood', 'Swan Valley', and 'Tuscola'
and one pinto bean (UI-lll) were germinated and grown in the green-

house in 30 x 55 cm plastic flats containing commercial vermiculite

(Terralite, W. R. Grace and Co., Cambridge, Massachusetts). Supple-
mental lighting from cool white fluorescent lamps provided an approxi-
mate 14 hour photoperiod. Nutrient levels were maintained with
weekly applications of 20N-20P-20K liquid fertilizer (Peter's Pro-
fessional, Peter's Fertilizer Products, W. R. Grace and Co.,

Cambridge, Massachusetts).

In,Vitro Culture Methods

Terminal shoot tips were harvested with a scapel from bean
plants at the primary leaf stage, usually 2 to 3 weeks old. These
initial shoot tips measured 1.0 to 1.5 cm in length and were surfaced
sterilized for two, 15 min. periods in a solution consisting of 15% ‘
commercial bleach and 0.01% sodium laurylsulfate. After they were
sterilized, the meristems were rinsed 6 times in double distilled
sterile water.

The nutrient medium used consisted of mineral salts
(Murashige and Skoog, 1965) with 0.8% Difco Bacto-agar and 3% sucrose.

11

12

TABLE 2.--Cu1tivars of common bean used in tissue culture experiments.

 

 

Commercial

Cultivar Class Seed Source Growth habit*

Fleetwood Navy Ontario I
Department of
Agriculture

Swan Valley Navy Michigan State II
University

Tuscola Navy Michigan State I
University

UIlll Pinto University of III
Idaho

 

*Growth habit designations were according to types defined by Centro
Investigaciones Agriculturo Internacional de Tropical (CIAT).

13

Vitamin supplements were as fbllows: 1.0 mg/l thiamine HCl and 0.5
mg/l each of nicotinic acid and pyridoxine HCl. To evaluate the
effect of different growth regulators and organic substances, benzyl-
aminopurine (BAP), kinetin, napthaleneacetic acid (NAA), indole
acetic acid (IAA) and casein hydrolysate and yeast extract were added
in a number of combinations and concentrations (Table 3). The pH
of all media tested was adjusted to 5.95 with 1.0 to 5.0 N KOH or
0.1 to 1.0 N HCl and then sterilized in a steam autoclave for 20 min.
at 15 p.s.i. (120°C).

Initial explants (apical buds) used in all experiments, were
cultured in 60 x 15 nnlstyle petri dishes containing approximately
10 m1 of nutrient media. Subsequent clones (ramets), derived from
the initial explants, were transferred after 3 to 4 weeks onto 30 to
35 ml of fresh media in 100 x 20 mm Corning petri dishes. In some
experiments where multiplied shoots were rooted, large glass jars
(Figure l) with 100 m1 of media were used. Unless indicated, all
cultures were maintained at a constant temperature (24°C) under a
16 hour photoperiod, maintained with fluorescent lights. Irradiation
just above the plants was measured with a quantum/radiometer/photo-
meter (Lambda Instruments, LI—185). The average light intensity

used was 19.0 11Em'2 sec'].

Explant Evaluation System

 

An evaluation system based on the number of shoots per
explant, callus and root formation and the overall vigor of each

initial and subcultured explant was used to select the optimal

14

 

Tissue culture derived plant
(Po Plant)

<l>

Po Seed

 

 

 

l P0 seed grown out to produce P1 plants

<l>

Seed

 

 

P1

 

| P.I seed grown out to produce P2 plants

P2 Seed

 

 

Figure l. Terminology used for seed derived from tissue culture
propagated plants. P = seed from plants cloned jg_vitro.

15

TABLE 3.--The major organic substrates and their concentrations used
in shoot multiplication and rooting media.

 

 

Chemical Range of

ingredient concentrations tested

Benzylaminopurine 0.1 to 100.0 mg/l
(BAP)

Casein Hydrolysate 0.0 to 400.0 mg/l
(CH)

Indoleacetic acid 0.05 to 0.6 mg/l
(IAA)

Napthaleneacetic acid 0.01 to 0.6 mg/l
(NAA)

Yeast Extract 0.0 to 400.0 mg/l
(YE)

Ammonium phosphate* 17.4 mM
(NH4H2PO4)

 

*Compound was the only component of the nutrient solution used to
fertilize transplanted bean plantlets. The ammonium phosphate was
dissolved in distilled water.

l6

compositions for shoot propagation and rooting media. The points
which were a part of the overall Vigor rating were: 1) primary
trifoliate leaf development, 2) color of the leaf tissue, chlorotic
versus green, and 3) multiple budding or branching of the explants.
In addition, the average leaf number and leaf area per explant was
estimated by subsampling 12.!1219 populations and counting the number
of leaves and measuring their areas with a square centimeter area

meter (Lambda Instruments, Model LI-3000, LI-COR).

Acclimation of Plantlets to Soil Environment

 

Plantlets were acclimated to potting soil conditions after
sufficient shoots and roots developed. Unless indicated all clones
were potted in a 1:1:1 (v/v/v) sterile mixture of vermiculite, com-
mercial potting mix (Metro mix, W. R. Grace and Co.) and soil. The
potting procedure is described as follows:

rooted plantlets were removed from the media and agar
clinging to the roots was washed away. Each planlet
was placed in a 50 ml styrofoam cup filled with approxi-
mately 150-200 grams of sterile soil mix that was
previously moistened with sterile distilled water and
fertilized with 30 ml of a nutrient solution (Table 3).
All pots were then covered with Saran Wrapc>(Dow
Chemical Co., Midland, Michigan) and incubated with
an irradiance of 48.0 pEm-Z sec-1, under a 16 hour
photoperiod of 25°C. After a time period ranging from
7 to 10 days, the potted plants were shaded under a
greenhouse bench for an additional 7 to 14 days.

After this, the Saran Wrap was removed and the pots
were placed in sealed high humidity chambers in full
sunlight for a period of 4 to 7 days. The tops of
each chamber were gradually opened after one or two
days to expose the plants to less humid air, thus,
adapting them to ambient greenhouse conditions. The
moisture level of each pot was periodically checked
during the acclimation period. If necessary the plants
were watered. When the plants became root bound they
were transplanted into 15 cm or 20 cm clay pots filled

17

with a previously moistened 1:1 (v/V) vermicultie to

soil mixture. All transplants at this stage were

fertilized weekly with a liquid fertilizer applica-

tion of 20N—20P-20K (Table 4).

A number of tissue culture derived plants were planted in the
field for evaluation. These plants were transplanted in the late
afternoon, shaded with dark screen for 3 days and then watered daily
for 1 and 1/2 weeks. After the bean transplants were fully adapted
to field conditions they were fertilized weekly with a 20N-20P-20K
liquid fertilizer. Some of the transplants were trellised with

4-mm - diameter cotton rope and stakes to avoid wind damage.

Field Experiments

 

Seed was collected from 14 Tuscola and 11 Swan Valley tissue
derived clones and designated Po seed (Figure 1). The P0 seed and
seed of two controls were grown for evaluation in a nursery at East
Lansing, Michigan in 1983. The controls were Tuscola and Swan Valley
plants grown from normally propagated seed. Rows were 150 cm in
length and spaced 50 cm apart. TWenty seeds per row of each entry
were planted and observations made on plants for anomalies in growth
habit and germination percentage. Similar evaluations were performed
on Tuscola and Swan Valley plants grown from tissue culture derived
seed which had been increased for one generation (East Lansing,
Michigan, Summer of 1982). This seed was designated P1 seed (Figure
1). Three entries of each cultivar and the two controls were grown
in three 50 foot long row plots spaced 50 cm apart. Fifteen grams

of seed (approximately 80 seeds) per row was planted. The experiment

18

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sprocaso

 

rug:

 

.coaaaeapuua Pram Lac acotatucou--.a msm<c

19

was replicated twice. In this experiment, flower fertility was
examined by sampling 10 plants per row and then counting the number
of pods and flowers formed per plant and comparing them to the
controls.

In a separate experiment, 23 UI-lll and 7 Fleetwood tissue
culture plants were placed in the field and observations on survival,
plant height, male fertility and pod-set were made throughout the
growing season. Transplant ratings for each transplant, ranging
from 0 (dead) to 5 (vigorous), were made 7 days after the initial

transfer to the field.

Characteristics of Tissue Culture Plants

Whole plants derived from tissue cultured explants were
rooted and grown to maturity in the greenhouse and evaluated for
morphological characteristics by counting the number of pods per
plant and examining leaf shape and size. Pollen fertility was
estimated by staining pollen grains with aceto-carmine and counting
the number of plump and evenly stained pollen grains which were taken
as fertile. The regularity of meiosis leading to normal fertility
was examined and estimated by counting the number of pods and seeds/
pods produced by each clone. Cytological analysis 0f.ifl.!i££9
propagated bean chromosomes was attempted but was unsuccessful
because of their extremely small size and the difficulty of

isolating and fixing chromosomes in the correst phase of meiosis.

20

Statistical Analysis

In the evaluation of the various growth regulators used in
this investigation, the appropriate analysis of variance were per-
formed and the means of different treatment effects within each
experiment conducted were compared by the use of Tukey's procedure
or t-tests. When a number of treatment combinations were found to
be non-significant, the selection of optimal treatment combinations

was made on the basis of subjective evaluation.

21

Figure 2.--Stages of the shoot multiplication system.

COW)

Source of apical buds
Stage 1 initial shoot-tip in petri dish
Initial and subcultured explant

Stage 2 subcultured multiple shoots in larger petri
dish

Stage 3 plantlets in larger vessel for expansion of
roots, leaves and stems.

22

_
“
a
n
“
TI.
E
E
IIL
rrl

‘.

 

23

 

24

 

25

Figure 3.--Stages of soil acclimation procedure.

A. Stage 1 rooted explant, prepared for potting.

B. Stage 2 plantlets placed into styrofoam pots, covered
with clear plastic film.

C. Stage 3 fully acclimated bean plants.

0. Field grown UIlll tissue culture plant.

26

 

27

 

28

 

RESULTS

Initial Studies

 

Yeast extract and casein hydrolysate at 0, 200 and 400 mg/l in
the basal medium along with BAP (2.0 mg/l) and NAA (0.1 mg/l) were inef-
fective in eliciting a response in the number of shoots or vigorous
growth of the bean explants. The use of yeast extract and casein hydro-

lysate was not considered for further bean tissue culture experiments.

Cytokinin Concentration Gradients

 

Concentration gradients with varying levels of kinetin (0.0
to 150.0 mg/l) and BAP (0.0 to 100.0 mg/l) caused a wide range of fin
:XiEEQ bean growth responses (Tables 5 and 6a, b; Figures 4, 5 and 6).
Nearly all concentrations of BAP gave a higher production of shoots
than the kinetin levels examined. Bean shoots responded to BAP at
3.0 mg/l by producing almost 6.0 subculturable shoots per explant;
whereas the optimal concentration of kinetin was 5.0 mg/l, producing
a mean of 4.4 shoots per explant (Figure 4). At higher concentrations
of BAP and kinetin (> 10.0 mg/l), shoot production as well as inter-
node elongation decreased sharply and rosette-like cultures with
small multiple buds, thickened petioles and stunted or deformed leaves
were produced (Figure 7). Bean shoot-tips grown or concentrations
between 75.0 and 150.0 mg/l kinetin showed slightly less multiple bud
differentiation and more callus production than the higher levels of

BAP examined (Figure 8). Although these high cytokinin concentrations

29

30

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copuuacoca mappmo mamcmvos u ++ .Azm ucmpnxm\ms coopuoomv mapped mppuwp ago> u + .mzppmu o: u o««
<<H F\me m.o spas.

 

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.mm>amp Fpmsm zgm> .upuocum:
meow .mmpowuma umcmxupza .muzn mpgwupas

 

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---s--- -----_\ss----
mmcoamwc guzoco «swappmu acmuoom sa<m mo
cowpmcucwocou

 

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31

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.<<H —\ms m.c ;u_3 s

 

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sup: mmc:p_=u mxwp1muummoc .uuaasou +++ 1 o.o~
.mm>mmp Pposm cam mucoccmacw umcwpcocm

;u_z mataapsu uxwp1apaamot .muss apatapsz +++ 1 o.o_

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acmsaopm>mv opp»VP vca mean opawupas o:

 

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.muza
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32

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.<<H P\mE m.o saw: a

+ .mappmu o: u or;

 

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111x111 11111111P\ms1111
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we cowumcucwocou

 

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33

 

 

 

UI-11 1
i-
Z
S BAP H
% KINETIN ---o-
C
LIJ
O.
U)
I'-
o
o
I
U)
1.0 »
0.0 1.0 10.0 1000

CONC. IN mg/l

Figure 4. Effects of cytokinin concentration on shoot production of
UIlll apical buds.

BASAL CALLUS (mg)

5000

V

4000

T

3000 r

2000 r

1000

 

0.0

Figure 5.

 

34

Ul-111

BAP H
KINETIN """"

1.0 10.0 100.0
CONC. IN mg/I

Effects of cytokinin concentrations or basal callus
formation from UIlll apical buds.

Figure 6.

BASAL CALLUS

35

 

 

4000
.\
3000- «K
2000 r ‘u\
\\
\\\
1000 ~ K\
\ \.
0 1 . .
0.0 50.0 100.0 150.0

mg / liter KINETIN

FLEETWOOD

Effect of kinetin concentrations on basal callus forma-
tion from Fleetwood shoot-tips.

36

Figure 7.--Multiple buds which differentiated at a single axial of
a Tuscola shoot culture grown on 50.0 mg/l BAP.

37

 

38

 

39

Figure 8.--Morphologic responses of ig_vitr0 cultured UIlll and
Fleetwood apical buds grown on 75.0, 100.0 and 150.0
mg/l kinetin.

KINETIN mg/p‘l

FLEETWOC‘D Ul-lll, 'lflTO

KINETIN mg‘r‘l

9"»
_ 3,
\i‘,

FLEETWOOD

 

41

KINETIN mg/l

(l l 1"

FLEETWOOD UI-111, PINTO

 

42

produced a prolific number of multiple buds, few whole plants could
be recovered from them and most of the bud masses remained quite small
or died when they were transferred to a basal medium containing no
growth regulators.

Bean shoot-tips cultured on lower levels of BAP, between 0.3
and 3.0 mg/l and kinetin between 1.0 and 5.0 mg/l were vigorous, pro-
duced little callus and formed a large number of subculturable shoots
per explant. These cultures were more useful with regard to shoot-tip
propagation than the beans grown on the high cytokinin concentrations.
Bean shoots grown at high and low levels of BAP are pictured in
Figure 9.

The effect of increasing cytokinin concentrations on bean
callus formation was measured by the fresh weight of basal callus per
explant. The addition of kinetin to the media resulted in the produc-
tion of larger amounts of callus than BAP at the same concentration.
High levels of BAP, greater than 10.0 mg/l appeared to inhibit callus
production (Figure 5), as did concentrations of kinetin greater than

75.0 mg/l (Figure 6).

Cytokinin - Auxin Combinations

Bean shoot-tips responded to each of the cytokinin-auxin com-
binations (i.e., kinetin x NAA, kinetin x IAA, BAP x NAA and BAP x IAA)
conducted with different degrees of success. Mean squares, means and

jn_vitro growth responses for all cytokinin-auxin combinations are

summarized in Tables 8, 9, 10, 11, 12, 13, 14, 15 and Figures 10, ll,
12 and 13). The optimal media selections from each grid are listed

in Table 7. Combinations of BAP and NAA appeared to be the most

43

Figure 9.--A. Bean shoot-tips cultured on 0.3 mg/l BAP.

8. Bean shoot tips cultured on 30.0 mg/l BAP.

44

 

 

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46

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47

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48

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49

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54

Figure 10. Effects of kinetin, BAP and NAA on shoot formation, shoot
length and callus production of Fleetwood shoot-tips.

55

 

 

 

 

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56

Effects of kinetin, BAP and NAA on shoot formation, shoot
length and callus production of UI-lll shoot-tips.

 

 

57

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58

Figure 12. Effects of kinetin, BAP and IAA on shoot formation, shoot
length and callus production of Fleetwood shoot-tips.

 

 

 

 

59

 

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60

Figure 13. Effects of kinetin, BAP and IAA on shoot formation, shoot
length and callus production of UI-lll shoot-tips.

61

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62

successful for bean shoot-tip propagation. At 3.0 mg/l BAP and 0.1
mg/l NAA bean apical buds proliferated and produced slightly more mul-
tiple buds and shoots than any of the other concentrations of BAP and
NAA tested. After 30 days from the initial culture, each shoot-tip
differentiated into highly branched plantlets with multiple buds which
ranged from 2 to 10 mm in length at each internode or leaf axial.
Adventitious buds arising from callus were frequently observed on this
media (Figure 14). Profuse callus production from the base of each
shoot-tip occurred with all combinations of BAP and NAA. Callus pro-
duction was greater in the BAP x NAA grid than in any of the others
conducted and no root formation occurred at any of the BAP or NAA levels
used. Shoot elongation was substantial (25 to 50 mm) at nearly all con-
centrations of BAP and NAA, thus requiring little effort to make the
divisions and subsequent subculture of each bean plantlet to fresh
media (Figure 15).

The effects of different BAP-IAA combinations of in 11559 bean
shoot growth were much like those found with BAP and NAA. However,
the optimal combination of BAP at 1.0 mg/l and IAA at 0.05 mg/l pro-
duced bean plantlets with shortened internodes and fewer multiple buds
and shoots (Figure 15). In comparison to the BAP-NAA combinations
examined, less basal callus was produced at all levels of BAP and IAA.
In addition, no root development or adventitious bud formation from
callus occurred. The shortened internodes and rosette-like structure
of these cultures resulted in some difficulty in dividing and sub-

culturing the newly formed shoots and buds on to fresh media.

63

 

Figure 14. Adventitious buds arising from callus of Fleetwood
clones cultured on 3.0 mg/l BAP and 0.1 mg/l NAA.

64

Figure 15. A. Bean plantlets cultured on BIZ (1.0 mg/l BAP and 0.05
mg/l IAA) and BN3 (3.0 mg/l and 0.1 mg/l NAA).

B. Bean plantlet cultured on BN3 (3.0 mg/l and 0.1 mg/l
NAA.

65

 

 

66

 

67

Both kinetin grids performed similarly and the most striking
feature of all kinetin-auxin combinations was the high frequency of
rooting and lack of multiple bud formation. Although the kinetin con-
centrations examined in this investigation produced only single shoots
and thus were not effective for multiple shoot production, kinetin
seemed to enhance lg gitgg bean leaf expansion and root production.

In the kinetin x NAA grid, significant genotypic differences in root
production were observed. The incidence of root formation of UIlll,
(indeterminate vine) was greater than that of Fleetwood (determinate).
Fleetwood rooted at a low frequency (10 to 40%) on only three combina-
tions of kinetin and NAA, whereas UIlll explants formed roots 20 to
90% of the time irrespective of the concentration of either hormone
(Table 16). Similar genotypic differences in root formation were

also observed in the kinetin x IAA grid (Table 17).

Of all kinetin-IAA combinations tested, kinetin at 3.0 mg/l
and 0.05 mg/l IAA was the best for shoot-tip pr0pagation. However,
when compared to optimal BAP-auxin levels, the multiple shoot forma-
tion capacity of kinetin and IAA together was poor. An unusual
phenomena was noticed in all kinetin-IAA combinations and that was
that further plantlet development beyond the primary trifoliate leaf,
i.e., shoot elongation and branching, was restricted from 20 to 100%
of the time (Table 18; Figure 16). This resulted in the formation of
explants with a single large leaf (primary trifoliate) which had no
potential for further propagation. When these leaves were subcul-
tured on to fresh media containing the same concentrations of kinetin

and IAA they frequently became necrotic and died.

6B

 

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69

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71

 

Figure 16. Primary trifoliate leaf development of bean shoot-tips
cultured on 3.0 mg/l kinetin and 0.05 mg/l IAA.

72

Final Media Selections

 

From each initial two-way cytokinin-auxin grid several opti-
mal hormone combinations were re-evaluated and four media combina-
tions were selected which were BN3, BIZ, KN3 and K13 (Table 7).

These media were compared over a number of subcultures (Figure 23)
and observations were made for; 1) the number of subculturable shoots
formed per explant, 2) the number of leaves per explant, 3) the
average leaf area per explant, and 4) multiple bud formation and root
development. Both BN3 and B11 media had a higher rate of shoot multi-
plication which was sustained for at least 100 days (5 subcultures

to fresh media) than that of KN3 and KI3 (Tables 19a, b; Figures 17
and 18). As the duration of culture on KN3 and K13 increased bean
shoot production decreased sharply and less vigorous plantlets were
produced. A large number of leaves were produced by the bean shoots
cultured on either BN3 or BIZ, whereas shoots grown on KN3 or KI3
formed only a few large leaves (Figures 19 and 20). When the leaf
areas of the plantlets grown on each media were measured and compared,
the leaves of shoots cultured on BN3 and B12 were found to be sub-
stantially smaller than those formed on either KN3 or K13. Beans
grown on BN3 had leaves almost one-half the size of those grown on
KN3 or KI3. Similar results were observed with shoot cultures of

B12 (Figures 21 and 22). No root formation occurred at any sub-
culture when bean explants were grown on either of the BAP media.

0n the other hand, rooting of shoots was frequent (20 to 100% on

both KN3 and KI3 media (Table 20). Multiple bud and shoot formation

73

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75

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76

30-

EE

5 FLEETWOOD
O.

55 BNr-*F-0-
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00 I 20 40 6‘0 80 160—“
suocunune (DAYS)

Figure 17. Effects of media (BN3, BIZ, KN3, KI3) and number of
subcultures on shoot production of Fleetwood tissue
cultures.

77

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'1—
§
5
Lu BN: H
E Bl: "’--"l’
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0 20 4o 60 116 100
suecunune (DAYS)

Figure 18. Effect of media (BN3, B12, KN3, K13) and number of sub-
cultures on shoot production of UIlll tissue cultures.

7B

15

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(SUBCULTURE (DAYS)

Figure 19. Effect of media (BN3, BIZ, KN3, KI3) and number of
subcultures on leaf production of Fleetwood tissue

cultures.

 

 

79

 

 

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.SUBCULTURE (DAYS)

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KN: '*““-*"
'Kl: --A---A-

Effect of media (BN3, BIZ, KN3, K13) and number of sub-
cultures on leaf production of UIlll tissue cultures.

80

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SUBCULTURE (DAYS)

Figure 21. Effect of media (BN3, BIZ, KN3, KI3) and number of sub-
cultures on leaf area of Fleetwood tissue cultures.

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81

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82

was observed throughout the 100 day period in all cultures of BN3.
The BIZ media performed in a similar fashion, however, fewer multiple
shoots were produced as the culture time on this media was increased.
Single shoots with large leaves were always the product of both the
KN3 and K13 media. Typical explants derived from each shoot culture

media are illustrated in Figure 23.

Light

2

The three light levels examined (high, 40.0 uEM' sec’1;

2 l Zsec‘l) were found to only

medium, 15.5 uEm' sec' ; and low, 3.0 pEm'
influence the green coloration of the cultured beans and presumably
their chlorophyll content and not their shoot proliferation. Those
explants kept in very high light conditions had chloratic foliage

with some necrotic areas, whereas those grown in medium or low light

were dark green in color and appeared to be normal.

Ig_Vitro Rooting

 

In screening the different cytokinin-auxin combinations for
shoot production, six potential rooting media were identified.
These selections were chosen as optimum for jg_yjtrg bean root forma-
tion (Table 21). The basal medium with or without auxin (either
NAA or IAA) proved to be the most successful. The addition of

kinetin at 0.5 mg/l to the media did not enhance in vitro root forma-

 

tion. Leaf expansion and shoot elongation of the explants grown on
kinetin, however, was better than those grown in the absence of
kinetin. Root formation did not occur with any level of BAP examined

in this investigation.

83

Figure 23.-~Fleetwood bean shoots grown on BN3, BIZ, KN3 and K13 after
100 days in culture and 5 subcultures onto fresh media.

 

 

85

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86

Acclimation to Soil Environment

 

The final step in the propagation of bean plants by tissue
culture was to adapt the clones to ex 11339 soil and greenhouse
environments. Difficulties with insect pests and dessication were
initially encountered, but a humid, aseptic acclimation procedure was
developed to circumvent the problems (Table 4). None of the four
sterile potting media examined: sand, vermiculite, Metro mix or
vermiculite mixture significantly influenced the survival or vigor
of transplanted bean ramets. However, the leaves of bean plantlets
grown in vermiculite alone tended to dessicate when exposed to dryer
air and frequently these pots required more water than the others.
Two or three fertilizations with 30 m1 of ammonium phosphate (17.4
mM) immediately after transfer to soil, appeared to improve the
vigor and leaf coloration of the rooted clones, although no signifi-
cant differences between the non-fertilized and fertilized trans-
plants were observed. Fully acclimated tissue culture derived bean

plants are illustrated in Figure 24.

Field Experiments

 

The 30 tissue culture derived plants (23 UIlll and 7 Fleet-
wood) which were planted in the field grow very well. All of
these plants were acclimated to soil conditions 120 days after initia-
tion in tissue culture (i.e., plants were started in February, 1983 and
placed in soil by May, 1983). The survival rate of the transplants
was 90%. These remained healthy and grew vigorously throughout the

season to maturity. An analysis of male fertility was conducted and

Figure 24.

A.

87

Fully acclimated tissue culture derived Tuscola bean
plant nearing maturity.

Fully acclimated tissue culture derived Swan Valley
and Tuscola bean plants (3 months from initial cul-
ture .

 

89

a high percentage of stained pollen (fertile) was observed (Table
22). The number of stained pollen grains collected from the tissue
culture derived plants was comparable to that of the normal, seed
propagated plants used as checks (Table 22).

The percent germination and growth habit of plants grown from
P0 seed (Figure 1) was apparently normal, with only one of the four-
teen Tuscola entries showing low germination (40%, Table 23). The
germination of other Tuscola and, the Swan Valley entries examined
ranged between 85 and 100 percent.

Analysis of the flower and pod set of Tuscola and Swan Valley
plants grown from P1 seed (Figure 1) indicated that there were signi-
ficant differences between the tissue culture derived plants (P1 seed
plants) and the controls (normal, seed propagated plants) Tables 24
and 25). Bean plants grown from P1 seed flowered later and there-
fore had formed fewer pods than the controls at the sampling time.
The P1 seed plants otherwise appeared normal with respect to growth

habit and germination.

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91

TABLE 23.--Percent germination of seed obtained from In vitro pro-
pagated bean plants.

 

Percent Germination*

 

 

Entry Tuscola Swan valley
1 100 90
2 100 100
3 100 100
4 95 95
5 100 95
6 95 100
7 100 100
8 100 95
9 40 100

10 100 100

ll 95 100

12 85 ---

13 85 ---

14 90 ---

Control 50 100

 

*Exactly 20 seeds from each entry were tested, the percentages repre-
sent the number of plants per row two weeks after planting. Seeds
from 14 Tuscola and 11 Swan Valley clones, plus two controls of each
genotype were used.

92

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93

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DISCUSSION

The MS (1965) basal media with 3.0 mg/l BAP and 0.1 mg/l NAA
(BN3) was the most effective combination for the induction of multiple
bud differentiation from shoot-tip cultures of bean. These condi-

tions are similar to those reported by Kartha, et a1. (1981) in his

 

study of bean plantlet regeneration from shoot meristems. However,
in this study greater success with regard to shoot multiplication
and whole plant recovery was attained.

The results of this research showed that the cytokinin level
(BAP and kinetin) was important to promoting multiple buds from bean
shoot-tips. 0f the two cytokinins examined, BAP was the most useful
in producing multiple shoots. 0n the other hand, most of the kinetin
concentrations used stimulated only the formation of single shoots
(Tables 5 and 6a, b). In the initial screening of hormone concen-
trations it was shown that very high concentrations of BAP and
kinetin (greater than 10.0 mg/l) induced the formation of large
numbers of small multiple buds. However, the survival of these
shoot cultures was very poor and only lower concentrations of BAP

and auxin (IAA or NAA) could sustain long-term vigorous jn_vitro

 

bean shoot multiplication (Table 19b). In addition, the levels of
casein hydrolysate and yeast extract that were tested failed

influence jn_vitro bean shoot-tip development.

95

Media which promoted multiple shoot production (BN3) arrested
root development and vice versa. The frequency of root formation was
high when the bean plantlets were cultured on basal media with or
without kinetin (0.5 mg/l) and IAA (0.3 mg/l) or NAA (0.6 mg/l)

(Table 21). Similar results were obtained by Geneve and Heuser (1982)
in their study of the effect of IAA, IBA, NAA and 2, 4-0 on root promo-

tion and ethylene evolution in Vigna radiata L. R. Wilez cuttings.

 

They found that for adventitious root initiation NAA and IBA (10'7 to
10'3M) were the most effective. The induction of root differentia-
tion also occurred on basal media without any growth regulators.
However, the cultures grown on this media formed less vigorous root
systems than those grown on either NAA or IAA (Table 21).

No difficulty in rooting and acclimating tissue culture was
encountered when an aseptic and humid potting procedure was employed.
The rooted bean plantlets which grew to maturity in either the green-
house or field appeared to be morphologically and genetically similar
to their donor parents. Analysis of the male fertility of tissue
culture propagated UIlll and Fleetwood plants ranged from 85 to 97%
and was comparable to the corresponding controls which consisted of
normal seed propagated bean plants of the same genotype. The germina-
tion of seed generated from bean clones was apparently normal, with
only one of 14 Tuscola entries showing low (40%) germination. An
important point to note is that the percent germination of the control
(normal, seed propagated Tuscola plants) was also low (50%) however,
this poor germination may have been due to the use of inviable seed

(Tables 22 and Z3).

96

Tuscola and Swan Valley plants grown from P1 seed appeared
to be morphologically similar to the controls. However, significant
differences with respect to the numbers of flowers and pods set were
observed (Table 24 and 25). Shepard, et al., (1980) noticed that
several of his tissue culture derived potato protoclones had altered
photoperiod requirements. Similar changes may have occurred in this
investigation. The plants from which this seed was obtained may
have initially been derived from adventitious buds which formed in
the basal callus of shoot cultures, rather than from axillary shoots.
These differences may also be the result of disease, moisture and
insect pressures and further research in this area is required.

The difficulty of obtaining large numbers of crossed seed
and each of suitable genetic or cytoplasmic male sterility systems
has limited the application of recurrent selection programs to bean
breeding(Brim, 1973). Kenworthy and Brim (1979) stated that pro-
cedures to reduce the time required to complete each cycle of selec-
tion are needed to increase the use and efficiency of bean recurrent
selection programs. In_yitrg bean propagation is an alternative to
making many hand cross-pollinations to obtain sufficient hybrid
seed for use in recurrent selection and hence eliminates some of the
labor requirements inherent with this breeding technique.

An additional use of in 31559 bean propagation is long-term
storage of genotypes. Saunders (1982) cited the possibility of
storing sugarbeet shoot cultures at low temperatures to facilitate
crosses between superior combiners from different years. With the

proper environmental conditions bean shoot cultures could be

97

maintained from one year to the next with a minimum of cost and
space.

Based on the research conducted herein, future studies on
bean tissue culture propagation should determine the degree of
genetic variability associated with plants resulting from axillary
bud proliferation in contrast to those arising adventitiously. In
addition, this research provides important information concerning

growth and development in bean cell and tissue culture.

98

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