4.....Vfl

    
    
     

‘ , ‘ III;

1" '.-; “',' 3,633,353, Ig-I‘I'U. - I, s .. ...
II III‘HI IIIIIW -“ -
I 111111.» .-

   
  
 

      
  
 

 

fifiquIuyv Iuw mfignrnz '.rer
I? W
I

1 II I ‘ , (I, 3:; . , |
. III .1'IIII'III..11‘.II
I; IIII‘IJ'III,','3{R3III,IIIIe-'I,I' (“I IJI 1%,, II‘II U: IMIII 013
”WWMWMIMNWMIVMMWIMH
I I II

I
's‘~,_-11I111I.II. ..

    

   
    
   
 
     
 
 
     
    
   
 

 

-- -1 , I
[3.1 I I III III
III ’,‘,KII39IHII'II, I “TIEII‘I “II? :fiIIlH' IIIIIII,”

I I.“ I II'I I' 3311‘“)
“1” WII qu

I 3 I I I I
I |II II 11" II III " I I
‘13,. I ‘11,I,,II,I,IH IIIIlIl IIIII ,,,',II «4.1 ”1'.

'II" ”I . III III -.

, ,,I,I,I,I ”,1”, I,II,,III.I,1I 11.1 12/ I I

II'II.IIIII-IIIII’III'II- I '
1

     

7‘

‘
“
_‘_ :_
{:45

III, III" IIIIIII 'II I

IJII‘IIIII, III III'II’I' ”I”

II I111 I I'll, '1‘,

I II. IIIIIIIIIHIIIIIIII “I '
:11 IIIIIIII'IIII‘ 'I-IIII"'I'II”III‘I' ? ‘IIII'I’II ‘ ”III '
',' 1' II.) I',.‘ IIIIII'I 1 '1 IIIIIH, MI 1., '-
.U¢1.mmflhmww-» . . 4,
‘IIIIIII..IIIIIIIIlrI-.I141' . WWI" II“ II I I .
IIIIP”IIIIIIHIIIIH.“ZII w‘ I»I .1.
I,.: I,I.I ,Il, 4,541, ,I,, " .' I; ' ,1,
1111.751II'I‘,.'II,I'II.II,"II III ' I,.II.I1IIII'IU I'III' ‘ ,
I,I3IIII 1,.II'I‘. .|| .III,III'I,,,,II ,I,I'1.IIIII“III,IIII N25,, 2 .‘I.
. ‘ ' IIIII IIIHW MI IIII ,h ;;

‘I.I;,II I
3:

  

    
  

  

      
     
 
  

 

I .I, 03“
I51”.I* III. III 11 MIW ‘r«54. .'
,ll“.," IllIlIyIII.‘IIIIII.,I,‘,'.;III3II,,I'IIIIII:';II‘I1'I.:‘I;; , E:

I. In .III.I .uI. vu.I, IIHIIIH 1‘ - .gg,

,I , I ‘ I ,.I, .II 3. .'.'l'I 3 31. I!
,l,,I, ,, 1.,,I,,I I _, , I ., ,,I,3,,, INI II . ”III’ :33}:

1 ,I .. I I ,...I3.,I ' ' .,.I_ 1' ,3, .
I, I.I ',‘ II,.,I'I , I- 1,1,. ., IIIII‘I. I,‘ I I‘II.“ IIIIIIII‘ III d] $3.5
“I“ . , ,,.~I.1 "”‘I.I..II.'I"' 11:. 1"II ' III '7’ .II‘I 1’11.
’1' .. .‘ . .-. 1 "' II .,_ .11 I “'3."
.‘ ,I I . I, II‘ .‘ 1' 'I,,‘ “ , 'l. , ,\ .,L-I,I 'f,'- ‘1' I II) “II I: ‘1...
II. ‘.IIg,g.'-. .., ,, ,.. III IIIIIL: ...
3"" I‘I IIIIII -.III,:I'-II 'II'II‘III’II II 'II .I III-'Il‘ .- r.I'II“ II' 'I I.

. III ' II. ‘I I " III I" .I'1 .' 'I~I-vI-II' II" IIII' ‘11.“: -.

. . ‘ I... '.II"I'II .I I. .I I~I.I'I'1 ','.‘I‘. ;I f'I.' I I'I I .‘I I - . - . . :
”M , , 1,1 ,’I-' .' , '1‘ , III,I,,.I,II,I:|,II':13II,I'.I.II‘III‘IIIIIII'IJI:,fl'I-IIJII‘HII‘I,: .I,(II ' 3,. II;:‘:1..1.§IJ “ ‘ 3" I ‘1‘ 1’11.
.I ,I I, , , II I“ 'l‘!ll."‘vfi ‘: , .‘ ...
IIIIIIV”.IIII ‘ 1.,I,, ,I‘IM I‘"I I‘ IIH'I IIi In 1‘ II"II«1,0I I'II' "- ~'..; - ”I?
,. I. III ‘ I. 3, ,, . I I’ I 1'... ' o

I I'I‘III'II' ‘1 ‘. ' II‘,'1‘,- III I. I IIIIIIJI'I'IIIII.I1 “-III 1 IIIIIIII. 5'15. III,,' -'

I, 1,I,,‘,I‘I ,I II ,Ihjl,,.‘I'. , I ,III,I.'i 1:3,, , II.I.'.‘.II.I11III I I 1., 1 ,1 I III‘I'W .1 , ‘
I I I I HIII 'IIIII,IIIII,II',I,I LII I II,,1-‘III',‘,II‘I I I” I "13 IIII' II31,',IIII’I’II’IIII'"V :1:
:‘II . II; II I I I . .3 I’3'IIIII IIII II II
IIIIIII I'IIIIII' I'IIIIIII lIII IIIIII III IIIII III III I". III' LIII IIIII'IIIII IIIIII IIIII’
.I I , IHIIII “1,1,,le I”, I,,,,I" ,,,,, ,I,I,,, , ’11,,,,‘,I,, :IMITIIIIJJ' III ,II!,'III,I:I."{','-’53“)JIIpr'IigfI'IflII‘II

‘ I .. '. III I‘ . 1H1 II' I I -I'II.;-
I I I IIIII ‘ IIII” II £I " JII S "IH1““II' IICIIUIII' 'WIIIIIII
3III31II, ,,III 13, 3H,I,,I'I, ,1, I,“ , 3 3,, ,,,I.III, ,: ,3“ ,,||':'. 1 ,II’I I,jIIIIII: I:,/,(‘II,|III'3 {I ”31,3”? I,!

“1.11 I II' .-"I 1”,..1111' v I. ‘ ' 'I' III I...

""‘ I ‘ ..I . . I I? ..-.I I" ‘,'IIII' I-' ”I III'IIIIII-III'IIIIII'II"

IIIII I, I
l . ' ,, IIIII IIIIIII'I
I" . . ""‘III .‘- II: ‘IW'IIII’II

. 1;"
IHI HIHIIIH

IIlII ,IIIIII,«,

W“
‘3.-

 

~———

   
 

"HE‘SIS

LIBRA R V '
Michigan State:- I
University {J

 

_‘\

This is to certify that the

thesis entitled

HORMONAL CONTROL OF ORGANOGENESIS ON LEAF
EXPLANTS OF BROWALLIA

presented by

Kent James Welsh

has been accepted towards fulfillment
of the requirements for

 

M g degree in Jim—tare

W CM

Major professor

 

Date ”(/7/72

0-7639

 

 

 

OVERDUE FINES:
25¢ per day per item
RETURNING LIBRARY MATERIALS:

Place in book return to remove
charge from circulation records

 

 

HORMONAL CONTROL OF ORGANOGENESIS ON LEAF
EXPLANTS OF BROWALLIA

 

By

Kent James Welsh

A THESIS

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

MASTER OF SCIENCE

Department of Horticulture

1979

ABSTRACT

HORMONAL CONTROL OF ORGANOGENESIS ON LEAF
EXPLANTS OF BROWALLIA

By

Kent James Welsh

hormonal control of callus, adventitious shoot and
root induction on leaf eXplants of Browallia viscosa and

3. speciosa was determined. Leaf emplants were aseptically

 

cultured on M/S salts and vitamins to which the auxins
1AA, IBA, NAA and 2,4—D and the cytokinins 2 ip, K, DA and
Zeatin were added singly or in various combinations.

owallia viscosa reSponded readily in vitro and extensive

U?
H

callus growth occurred on several media at 1500 lux and
2536 :_2. Shoots arose when callus was cultured on m/S
plus 2 i;, K and EA, but only consistently on 2 ip. The
shoots rooted very easily in four to seven days in an arti-

ficial planting medium. Browallia Speciosa responded

 

slower in culture, but callus was initiated and maintained
on m/s plus 0.5 or 2.5 mg/l BA and 5.0 mg/l 2,4-D or on

Uchimiya and Murashige basal medium, U/M. The regenerated
plants of Q. viscosa appeared to maintain the morphologi—

cal and cytological traits of the parent.

To Laura Lee, Jennifer and Melanie

ii

ACKNOWLEDGMENTS

I would like to thank Dr. Kenneth C. Sink for his
assistance in this research program and in editing this
thesis. I would also thank Dr. Robert C. Herner and Dr.
Harry H. Murakishi for their help in the preparation of
this thesis. Thanks and appreciation also go to my parents
for the upbringing and encouragement they have given to
me. My wife also deserves thanks for her support, inspir-

ation and love during time spent writing this thesis.

iii

TABLE OF CONTENTS

LIST OF TABLES 00.0.0000...OOOOOOOOCOOOOOOOOOOOOOOO Vi

LIST OF FIGURES 0.0.0.0.0.000000000000000000000000. Vii

 

IIITRODUCTIOIQ O O O O O I O O O O I O O O C O O O O I O O O O O O O O O O O O O O O O O 0
LITERATURE REVIEVV O O O O O O O O O O O O O O O O O C O O O O O O O O O O O O O O O
IVIATERIALS MID IVIETHODS O O O O O O O O O O O O O O O 0 O O O O O O O O O O O O 0

Plant material ...............................
Sterilization procedure ......................
Media no...co000000000oooooooooooooooooooooooo
Cytological studies ..........................
Pollen viability .............................

CMQUDGFJ -4 l» H

I4 H
[—4

RESULTS OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOCOOOO

Morphogenetic reSponse of leaf eXplants to

auxins ....................................... ll
Morphogenetic response of leaf eXplants to

cytokinins ................................... l3
Morphogenetic reSponse of callus to cyto-

kinins ....................................... 18
Morphogenetic response of leaf eXplants to
cytokinin/auxin combinations ................. 23
Shoot growth ................................. 28
Cytology and morphology of regenerated

plants ....................................... 29

DISCUSSIOIJ .0.0I..00...I...OOOOOCOOOOOOOOOOOOOOOOOO 34

Cultivated vs wild species ................... 34
Taxonomic differences ........................ 36
Cytological differences ...................... 37
Auxin specificity ............................ 38
Cytokinin specificity ........................ 4O
Cytokinin/auxin specificity .................. 41
Rooting ...................................... 43
Cytology-morphology of regenerated plants .... 43
Conclusions .................................. 44

iv

SUI‘H'IEARY 0.0.0....0..OOOOOOOOOOOOOOOOOOOOOOOO0.0...O

Browallia viscosa ............................

 

BrowalliaSJeCiosa OOOOOOOOOOOOOO0.00.0.0.0...

BIBLIOGRAPHY

45

45
46

4'7

LIST OF TABLES

Table l. Auxins and cytokinins and their
abbreViations 0.0.0.0....OOOOOOOOOOOOOOOOOOOOO 9

Table 2. The morphogenetic response of leaf ex—
plants of two Browallia Species cultured in
vitro for 6 weeks on M/S basal medium con-
taining zuxins ............................... l2

 

Table 3. The morphogenetic reSponse of Browallia
viscosa leaf eXplants and callus cultured
on basal medium containing cytokinins ........ l4

 

Table 4. Frequency of regenerated shoots on
Browallia viscosa leaf eXplants and callus
cultured on basal medium with cytokinins
for 6 and 12 weeks reSpectively .............. 19

 

vi

LIST OF FIGURES

Figure l. Adventitious shoot initiation on leaf

sections of Browallia viscosa cultured on

 

M/S basal medium plus 2 ip after 6 weeks in

culture 0..0......-OOOOOOOOIOOOOOOOIOOOO

Figure 2. Morphogenetic reSponse of leaf sec—
tions of Browallia viscosa to BA+NAA com-
binations after 6 weeks in culture .....

 

Figure 3. Morphogenetic response of leaf sec-

00....

tions of Browallia gpeciosa to BA+NAA com-

 

 

binations after 6 weeks in culture .....

Figure 4. Root develOpment on regenerated shoots
of Browallia viscosa cultured on M/S basal

 

medium plus IAA after 1 week in culture

vii

16

24

26

3O

INTRODUCTION

The Solanaceae or Nightshade family is composed of

 

several important economic genera as well as many which

are less important. Included in this family are the genera
Nicotiana (tobacco), Petunia, LIEOpersicon (tomato),
Solanum and others. An important ornamental genus of this
family is Browallia. There are four Species within this
genus, two of which, §._§peciosa Hook and g, viscosa HBK,
were chosen for study since they are ornamental types

used as bedding plants and for hanging baskets. They are
grown for their profuse masses of white or blue flowers
which continue blooming throughout the summer.

The Solanaceae are also important since many species

 

of this family can be manipulated in vitro for experi-

mental purposes. Browallia, however, has received little

 

or no attention and for this reason was selected for study.
This research was conducted to gain information on the
morphogenetic responses of leaf sections of g. speciosa

and g, viscosa cultured ig'zitgg on basal medium contain-
ing auxins or cytokinins and in combination. The main
emphasis of the research was to determine: 1) the environ-
mental and ig.3l£gg culture procedures for callus, root

and shoot initiation on leaf explants, 2) the medium

1

2

composition for shoot regeneration from callus cultures
and 3) the chromosome stability of regenerated shoots and

intact plants.

LITERATURE REVIEW

Tissue culture provides an eXperimental system for
the study of morphological responses of plant parts under
rigidly defined conditions. Some of the earliest work was
done by White (1934) working with tomato roots. By adding
yeast to his medium, he was able to grow tomato root tips
for unlimited lengths of time. The wild carrot, Daucus
carota L., also provided an excellent source of material
for in 33339 research. Other early workers (Steward et. al.,
1958; Reinert, 1959; Halperin and Wetherell, 1964; Halperin,
1966; and Linser and Neumann, 1968) conducted experiments
using roots, petioles and umbellate peduncles of this
Species. They were able to regenerate shoots and embryos
from these explants. Considerable 22.21232 studies have

been done on the Solanaceae. Several genera, Nicotiana

 

(Skoog and Tsui, 1948; Skoog and Miller, 1957; Gupta et.
al., 1966; Walkey and Woofitt, 1968; and Uchimiya and
Murashige, 1974) and Petunia (Handro et. al., 1972; Rao
et. al., 1973 a,b; and Swamy and Chacko, 1973) have been
studied in depth, whereas, others, Datura (Guha et. al.,
1964; and Engvild, 1973), Phxsalis (Zenkteler, 1973),
'Lycopersicon (White, 1934; Norton and B011, 1954;
Padmanabhan et. al., 1974; and Ohki et. al., 1978),

3

4

Salpiglossis (Hughes et. al., 1973; and Lee et. al., 1977).
Solanum (Rao and Narayanaswami, 1968 and Zenkteler, 1972)
and Browallia (Power and Berry, 1979) have received less
attention.

The genus Nicotiana, especially N. tabacum L. has been
used extensively to gain a great deal of information on
the control of morphogenesis and differentiation ig‘zitgg.
Skoog and Tsui (1948) found that adenine would induce bud
formation in callus and on stem internode segments ofgfi.
tabacum. They also found that NAA stimulated callus growth
and root formation. Adenine plus NAA was shown to cause
cell proliferation but did not cause bud formation. In
1966, Gupta et. al. were able to induce the differentiation
of adventitious buds on leaves of this Species.

Other Species within this genus have also been ex-
amined. Walkey and Woolfitt (1968) reported that callus
and adventitious Shoots developed from Shoot meristems of
‘N. rustica.

Petunia has also been studied extensively. The in
.XEEEB response of P. hybrida Hort. and P. inflata R. E.
Fries leaf and stem segments was determined by Handro
et. a1. (1972) and by Rao et. a1. (1973 a,b). They observed
callus, embryo, shoot, and root formation when different
auxins and cytokinins were added to M/S medium.

Swamy and Chacko (1973) also found that anthers of
‘3. axillaris (Lam) B.S.P. would develOp callus and plant—

1ets depending on the growth conditions.

5

Besides Nicotiana and Petunia, a number of other
genera in this family have been studied. Callus, adven-
titious Shoots and embryos have been reported from anthers
(Guha et. al., 1964) and stem segments (Engvild, 1973) of
Datura innoxia Mill. Norton and B011 (1954) observed shoot
and callus formation from roots of a clone of Lycopersicon
ppgruvianum (L.) Mill. Plantlets have also been induced

on callus generated on explants of L, esculentum

 

(Padmanabhan et. al., 1974). Other researchers have ob-
served callus and adventitious shoot formation from anthers
(Huhges et. al., 1973) and leaf sections (Lee et. al.,
1977) of Salpiglossis sinuata L. Zenkteler (1972) reported
the formation of buds on leaves of Solanum dulcamara L.,
S, nigrum L. and Physalis peruviana. These buds were
capable of develOping into shoots and plants. Power and
Berry (1979) observed shoot formation from callus grown
from leaves of g, viscosa HBK.

The response of a plant tissue in 31332 is mainly
controlled by the medium composition and environmental
conditions. And, it is clear from the work of Skoog and
Tsui (1948), Steward (1958), Skoog and Miller (1957) and
others that plant hormones, notably auxins and cytokinins,
at least partially, are responsible for the type of growth
which occurs. Of course not all plant species respond in
the same way to identical hormone treatments but there
are certain similarities in the types of response which

occur. Auxins generally promote callus growth and/or

6

rooting while cytokinins seem to be required at least in
some species for shoot initiation. Combinations of cyto-
kinins and auxins produce a variety of reSponseS from
callus and root induction to shoot initiation depending
on the hormones employed, their concentrations and the

plant Species under study.

MATERIALS AND METHODS
Plant material:

Two Species of Browallia, B. speciosa Hook cv. 'Major'
Blue Bells Regular and B, viscosa HBK., were used in this
study. Browallia speciosa is native to Columbia. Plants
of this species may attain 152.4 cm in height in natural
habitats and have large flowers 3.8-5.1 cm across. The
flower colors are blue, violet and white in selected vari-
eties. Browallia viscosa is common in Peru and other
South American countries. The flowers of this species
reach a diameter of 1.9 cm and are usually blue with a
yellow-white throat; although white flowered cultivars
also exist. Browallia viscosa generally grows to a height
of 30.5 cm. Extensive, detailed descriptions of these
two Species can be found in Hortuslghigg (1976).

Plants for igwxitgg studies were obtained from seed
sown on a weekly basis. Seed was sown on a moist artifi-
cial planting medium; the seed trays were covered with
clear plastic and placed under artificial continuous fluor-
escent light at 2600 lux at ambient room temperatures
(25 :_2°C).

After two to three weeks, the seedlings were trans-

7

8

planted to plastic trays in the greenhouse using an arti-
ficial planting medium. The seedlings were grown under
natural light conditions supplemented with extended light-
ing, 100 lux during the night from 10 P.M. to 2 A.M. The
night temperature was held at a minimum of 22°C. Fertili-
zer was applied to the plants twice per week using 20-20-
20 at the rate of 200 ppm N. The plants were watered as
needed and standard cultural practices were used to con-
trol Various insects.

Leaves for in 21339 culture were obtained from vege-

tative seedlings of B, viscosa and B. speciosa when they

 

were 40-60 and 60—80 days old reSpectively.

Sterilization procedure:

A laminar flow hood was used for all sterile proce-
dures. Leaves were surface disinfected by immersion in
four percent commercial sodium hypochlorite solution plus
0.02 percent Tween 20. After 30 minutes, the diluted
bleach solution was removed and the leaves were washed
with five separate sterile water rinses.

Disinfected leaves were prepared for culture by slic—
ing them into sections, 1/2-1 cm2. During this procedure,
the leaf margins and midrib were also removed. All the
eXplantS were subcultured to fresh medium every four weeks.
The cultures were held at 25°C under continuous cool white

fluorescent light at 1500 lux.

Media:

Murashige and Skoog (1962) salts and vitamins (M/S)
was generally used as the basal medium. Uchimiya and
Murashige (1974), (U/M) medium was also used to a limited
extent. Various auxins and cytokinins as described in
Table l. were added singly or in combination to the basal
medium. The growth regulators were added to the defined
medium prior to autoclaving, except for IAA which was
filter sterilized through a 0.22 Millipore filter and
aseptically added to cooled medium (40—4500) after auto-
claving. Sucrose at 3% and 0.8% agar were routinely added
to the M/S basal medium. The pH of all media was adjusted

to 5.8 prior to autoclaving at 15 psi, for 20 minutes.

Table l. Auxins and cytokinins and their abbreviations.

 

 

Auxins Bytokinins

Indole-acetic acid, IAA 6-furfury1amin0purine, K
Indole-butyric acid, IBA 6-benzylamin0purine, BA
Napthalene acetic acid, NAA 6-(tranS-4-hydroxy-3-methyl-
2,4-dichlor0phenoxyacetic but-2-enylamino) purine, Z
acid, 2,4-D 6(v-Vdimethyally1amino)-

purine, 2 ip

 

Bytological studies:

Shoot cuttings taken from seedlings of B._§peciosa,

 

B, viscosa and from regenerated plants were dipped in 0.3%

IBA and placed in perlite. The cuttings were placed on

10

an intermittent mist bench in the greenhouse where B,

Speciosa and B. viscosa rooted in ten and Seven days re-

 

Spectively. Roots 5—10 mm in length were removed, prefixed
in saturated ABN (c-bromonaphthalene) for two hours in the
dark at room temperature and fixed in 3:1, absolute a1-
cohol:glacial acetic acid (Sink and Power, 1977) overnight.
Fixed roots were stored in 70% ethanol at 4°C.

Roots were subsequently hydrolyzed in l N HCl at 60°C
for ten to fourteen minutes, placed on a Slide and smeared
with 1% acetocarmine stain (Darlington and La Cour, 1975).
A coverslip was placed over the cell smear and the slide
was gently heated. Following heating, the Slide was tapped
lightly with the blunt end of a wooden dissecting needle
to flatten the cells and the excess stain was removed.
Chromosome number was determined by counting a minimum of

eight good root tip cell preparations.

 

Pollen viabilit':

The pollen fertility of the Browallia Species and

 

the regenerated plants was determined by placing a dehisced
anther in a drop of 1% aniline blue (Johansen, 1940) on a
micrOSCOpe slide. The anthers and debris were removed and
a coverslip added. Excess stain was removed from the
slides and they were examined after standing for 20—25

minutes at room temperature for viable pollen.

RESULTS

Mogphogenetic response_gf leaf eXplants'Bg auxins:

 

 

The response of B. Speciosa and B. viscosa leaf sec-

 

tions on Murashige and Skoog (1962) (M/S) basal medium
with auxins incorporated is Shown in Table 2. Leaf sec-
tions of B. viscosa developed extensive roots when placed
on M/S+IAA, IBA or NAA.

0n the M/S+IAA media there was a gradual increase in
the number, length and degree of branching of roots as
the level of IAA increased from 0.01 mg/l to 5.0 mg/l and
at 10.0 mg/l they decreased. At 0.01 mg/l IAA, one or two
small, branched roots were observed per eXplant. Numerous,
well develOped and highly branched roots were observed at
5.0 mg/l IAA. Less root develOpment occurred at 10.0 mg/l
IAA. The roots which develOped at all IAA levels were
white and branched; callus growth was not observed.

After 6 weeks of culture, differences in root initia-
tion between the IBA levels were not distinguishable. Num-
erous, fine white, branched roots were observed in each
culture.

Leaf sections placed on M/S+NAA at 0.01—1.0 mg/l all

responded Similarly. They were dark green, with numerous,

11

12

Table 2. The morphogenetic reSponse of leaf eXplantS of
two Browallia Species cultured lg vitro for 6 weeks
on M/S basal medium containing aux1ns.

 

 

Auxin Conc. B, speciosa ‘B. viscosa
mg/l

 

 

MS

MS+IAA 0.01 N/T R
0.1 N/T R

1.0 N/T R

5.0 N/T R

10.0 N/T R

RS+IRA 0.01 — R
0.1 — R

1.0 — R

5.0 - R

10.0 — R

RS+RAA 0.01 - R
0.1 - R

1.0 — R

5.0 — —

10.0 — _

MS+2,4-D 0.01 - -
0.1 - c

1.0 — c

5.0 - C

10.0 - _

 

-, no response; 0, callus; R, roots; N/T, not tested.

13
highly branched, white roots. All leaf explants were dead
after 6 weeks when cultured at the two highest NAA concen—
trations. Very minute quantities of light brown callus
were observed at the cut edges of leaf pieces cultured on
IDA and NAA.

2,4-dichlor0phenoxyacetic acid (2,4-D) evoked a dif-
ferent morphogenetic reSponse than the other three auxins.
Leaf sections of B. viscosa had little or no callus for-
mation at 0.01 mg/l 2,4-D, increased to large amounts at
0.1 mg/l 2,4—D and decreased in quantity at the higher
concentrations. At 10.0 mg/l 2,4-D no callus production
occurred. The callus initiated on 2,4—D was yellow-white,
soft and friable in texture.

Leaf sections cultured on M/S basal medium without
auxins gave no reSponse or very infrequently produced
roots.

Browallia Speciosa did not reSpond to auxin supple—
ments. After 4 weeks in culture all of the leaf sections
were dead regardless of auxin type or concentration, except

for IAA which was not tested.

Morphogenetic reSponse.gf leaf eXplantS'tg gytokinins:

 

Callus or callus plus shoots were initiated on leaf
sections of B, viscosa when cultured on.M/S basal medium
containing the 3 cytokinins: K, BA or 2 ip (Table 3).
Most leaf explants plated on K or BA died within 6 weeks.

14

Table 3. The morphogenetic reSponse of Browallia viscosa
leaf eXplantS and callus cultured on basal med1um
containing cytokinins. Observations taken 6 and 12
weeks after plating the leaf eXplantS and callus re—
Spectively.

 

Cytokinin Conc. Leaf Sections Callus initiated on 2,4-D

 

mg/l 0.1 mg/l 1.0 mg/l
MS - - C C
MS+K 0.01 — 0 0,3
0.1 - 0,3 0,3
1.0 0 0 0,3
5.0 0,3 0,3 0
10.0 C C C
MS+BA 0.01 - 0,3 0,3
0.1 — 0 0,3
1.0 - C C
5.0 C C C
10.0 0 0,3 0
m3+2 ip 0.01 — 0,3 0
0.1 0 0,3 0
1.0 0,3 0,3 0,3
5.0 0,3 0,3 0,3
10.0 0,3 0,3 0,3

 

—, no reSponse; C, callus; S, shoots and/or leaf primordia.

14

Table 3. The morphogenetic reSponse of Browallia viscosa
leaf eXplantS and callus cultured on basal me ium
containing cytokinins. Observations taken 6 and 12
weeks after plating the leaf explants and callus re—

Spectively.

 

Cytokinin Cone. Leaf Sections Callus initiated on 2,4-D

 

mg/l 0.1 mg/l 1.0 mg/l
MS — — C C
MS+K 0.01 - 0 0,3
0.1 - C,S C,S
1.0 0 0 0,3
5.0 0,3 0,3 0
10.0 C C C
MS+BA 0.01 - C,S C,S
0.1 - 0 0,3
1.0 - C C
5.0 C C C
10.0 0 0,3 0
MS+2 1p 0.01 — 0,3 0
0.1 0 0,3 0
1.0 0,3 0,3 0,3
5.0 0,3 0,3 0,3
10.0 0,3 0,3 0,3

 

-, no reSponse; C, callus; S, Shoots and/or leaf primordia.

l5

Occasionally, very small amounts of green callus were
observed at the cut edges of explants cultured on media
containing the three highest levels of K. Also, one small
Shoot was observed at 5.0 mg/l K. Small to medium quan-
tities of compact green callus were also observed on basal
medium containing BA at 5.0 and 10.0 mg/l. No shoot ini-
tiation was observed on M/S medium to which BA was added.
Shoot initiation and growth was consistent only
when 2 ip was employed as the cytokinin. The first Shoots
were observed after 4 weeks at 1.0 mg/l 2 ip. Within 6
weeks after leaf Sections were placed in culture, leafy
growth and Shoots were observed on more than 85% of the
explants on 1.0, 5.0 and 10.0 mg/l 2 ip (Figure 1). A few
green Shoots, 2 cm in height and leaf-like structures were
present at 1.0 mg/l 2 ip. A small number of roots were
observed on the callus and on Several of the shoots at this
2 ip concentration. Larger shoots, up to 5 cm in height,
some with flowers and well developed, branched roots were
observed at 5.0 mg/l 2 ip. At 10.0 mg/l 2 ip, Shoots and
leafy growth 2 cm tall were observed with very few roots
also present on the callus.

Callus growth was also initiated when leaf sections of
‘B. viscosa were plated on.M/S+2 ip. Callus growth in—
creased progressively from zero at 0.01 mg/l 2 ip to large
quantities at 5.0 mg/l 2 ip. Only medium amounts were
present at 10.0 mg/l 2 ip. The callus at all 2 ip levels

was compact, yellow—green and nodular.

16

Figure 1. Adventitious shoot initiation on leaf sections
of Browallia viscosa cultured on M/S basal medium plus
2 ip after 6 weeks in culture. Concentrations of 2 ip
from left to right: 0.0, 0.01, 0.1, 1.0, 5.0 and 10.0

mg/l.

 

 

1?

 

Figure 1.

18

The intensity of Shoot formation in relation to the
different cytokinins is presented in Table 4. Shoot ini-
tiation and growth occurred on leaf Sections at: M/S+5.0
mg/l K and 1.0-10.0 mg/l 2 ip. The number of Shoots ini-
tiated at 1.0 and 5.0 mg/l 2 ip was very Similar with less
Shoot formation at 10.0 mg/l. Shoot development did occur
at 5.0 mg/l K, but was not consistent.

Leaf sections of B, Speciosa were also plated on M/S+

 

K, BA and 2 ip, however, after 4 weeks in culture all of

the explants had died.

Morphogenetic reSponse of callus to cytokinins:

 

 

Induction of shoots was also observed when callus de-
rived from leaf eXplantS of B. viscosa was cultured on K,
BA or 2 ip (Tables 3 and 4). Callus initiated on 2,4—D
at 0,1 and 1.0 mg/l was used to conduct these cytokinin ex-
periments.

Most of the callus which originated on 0.1 mg/l 2,4—D
died following plating on M/S+K. Small to medium amounts
of yellow-brown callus were observed at each K level, which
subsequently turned brown and died. Shoot formation did
occur however at 2 K concentrations. At 0.1 mg/l K, one
explant of 4 was greenish-white with numerous, small, dark,
green leaves. These leafy growths were 2-3 mm high.
Shoots, 2-3 mm tall with well develOped leaves and flower

buds were observed at 5.0 mg/l K on one piece of callus.

1

\0

Table 4. Frequency of regenerated Shoots on Browallia
viscosa leaf eXplants and callus cultured on basal
medium with cytokinins for 6 and 12 weeks respectively.

 

Cytokinin Conc. Leaf Sections Callus initiated on 2,4-D

 

mg/l 0.1 mg/l 1.0 mg/l
MS - - - -
MS+K 0.01 — - ++
0.1 - ++++ +
1-0 - - +++
5.0 + ++++ -
10.0 - - —
MS+BA 0.01 - + ++
00]. "" "' +
1.0 - - —
500 "" - .-
10.0 - ++++ -
MS+2 ip 0.01 — +++ —
Ool "' ++ —.
1.0 ++ ++ . ++
5.0 ++ + ++++
10.0 + + +

 

—, no shoots; +, less thn 5; ++ 5—20; +++, 20—50; ++++,
>50.

20

The callus ranged in color from light brown to green-
ish-white at 0.01—1.0 mg/l K. Shoots and/or leaves were
also observed at these K levels. Shoots, leaves and small
amounts of callus were initiated at 0.01 mg/l K. The
shoots were green and up to 2 cm in height. Dark green,
leafy appendages and moderate amounts of callus were ob-
served at 0.1 mg/l K. At 1.0 mg/l K, large amounts of
compact callus, small leaves and 1.5 cm long shoots devel-
Oped. Only one eXplant initiated shoots at each K level.
Moderate amounts of greenish-white callus growth were ob—
served at 5.0 and 10.0 mg/l K, but no Shoot initiation
occurred.

Callus cultured on M/S+BA reSponded very Similar to
that placed on K. All of the callus derived from 2,4-D
at 0.1 mg/l was dead or dying after 12 weeks. At each BA
level, small to medium amounts of yellow—brown callus de-
velOped before death occurred. Shoots were initiated at
0.01 and 10.0 mg/L BA. Only one explant per treatment
deve10ped Shoots. Three green shoots, 4 mm tall were ob-
served at 0.01 mg/l BA, while numerous leaves and small,

5 mm shoots originated on BA at 10.0 mg/l.

Callus induced on 2,4-D at 1.0 mg/l and transferred
to M/S+BA continued growth. Large amounts of semicompact
to very compact, brown to greenish-white callus develOped
on the eXplants at each BA level. Leaves and shoots oc-
curred at 0.01 mg/l BA with only leaves present at 0.1

mg/l BA. In each instance, only one of four cultures

21

develOped Shoots.

Callus from 0.1 and 1.0 mg/l 2,4-D reSponded similary
when subcultured on 2 ip (Tables 3 and 4). Medium to
large quantities of yellowish-green, nodular callus de—
velOped at each 2 ip concentration. Semicompact callus
was observed at 0.01 mg/l 2 ip and became progressively
more compact as the level of 2 ip increased to 1.0 mg/l.
The callus on 1.0-10.0 mg/l 2 ip was very hard and compact.
As with leaf sections, shoot proliferation was most con-
sistent on M/S basal medium containing 2 ip. However,
there were differences in the number and type of Shoot
growth depending on the callus source and concentration
of 2 ip.

Shoots and/or leaves arose on 0.01—10.0 mg/l 2 ip
with callus derived from 0.1 mg/l 2,4—D. The highest num-
ber and size of Shoots occurred at 0.01 mg/l 2 ip and de-
'creased as the 2 ip level increased. Approximately 25
shoots, 2 cm in height and leaves were initiated on 2 ip
at 0.01 mg/l. Less than 10 Shoots and leaves occurred
at 0.1 mg/l 2 ip. Only leaves were initiated at 1.0—10.0
mg/l 2 ip. Shoot initiation occurred on one of four ex—
plants at 0.01, 0.1, 5.0 and 10.0 mg/l 2 ip. At 1.0 mg/l

2 ip, 50% of the eXplantS develOped shoots.

No shoots developed on.M/S+2 ip at 0.01 or 0.1 mg/l
when callus from 1.0 mg/l 2,4—D was used. Shoots and
leaves did occur, however at 1.0—10.0 mg/l 2 ip. At 1.0

mg/l 2 ip, 10—20 well developed shoots, 1.5 cm in height

22
and leafy growth occurred. These shoots were dark green
and appeared morphologically normal. Numerous, small leaf-
like structures and shoots were present at 5.0 mg/l 2 ip.
Over 50% of the cultures at 1.0 and 5.0 mg/l 2 ip develOped
shoots. The number of shoots decreased at 10.0 mg/l 2 ip
to less than five. Only one of 4 cultures at 10.0 mg/l
2 ip develOped shoots.

Callus from 0.5 mg/l BA plus 5.0 mg/l 2,4-D was sub-
cultured to M/S+Z at 1.0 mg/l to determine Shoot regenera-
tion potential. No Shoots were observed at this Z concen-
tration after 12 weeks of culture. The callus was green—
ish-white, compact and Slow growing.

Callus of B, §peciosa initiated on 0.5 or 2.5 mg/l BA

 

plus 5.0 mg/l 2,4-D was subcultured to M/S basal medium
with K, BA or 2 ip. Twevle weeks after subcultures were
initiated, the callus on all media was either dead or turn—
ing brown and dyinv. The exception to this response was
that callus obtained from 2.5 mg/l BA plus 5.0 mg/l 2,4-D
placed on 1.0 mg/l Z remained greenish-white, semicompact
and increased Slightly in quantity. A few Single roots,
less than 5 per culture, with numerous root hairs were

also present.

The only difference in reSponse between the two callus
sources was in the quantity of callus growth before death
occurred. Only small amounts of callus develOped on ex-
plants derived from 0.5 mg/l BA plus 5.0 mg/l 2,4—D plated

on K, BA or 2 ip regardless of concentration. EXplantS from

23

2.5 mg/l BA plus 5.0 mg/l 2,4—D initiated medium—large

quantities of callus on all levels of the three cytokinins.

Morphogenetic reSponse 2: leaf explants £2 auxin/cytokinin

 

 

combinations:

 

Leaf sections Of.§- viscosa and B, Speciosa were

 

plated on BA plus NAA media (Figures 2 and 3). The media
combinations included each growth regulator at five levels:
0.01, 0.1, 1.0, 5.0 and 10.0 mg/l. With both species, cal-
lus growth increased from the lowest level of each hormone
to the highest level; however, their rate of growth was

not the same.

Callus was initiated on leaf eXplantS of B. viscosa
cultured on all but the lowest BA+NAA combinations (Figure
2). Yellow—green, compact callus was observed frequently
and the largest quantity of callus growth was produced on
leaf pieces placed on 10.0 mg/l BA plus 10.0 mg/l NAA.
Roots did not develOp on BA plus 0.01 or 0.1 mg/l NAA com—
binations. However, many white, branched roots were ob—
served on 0.01, 0.1, 1.0 mg/l BA on 1.0, 5.0 and 10.0 mg/l
NAA. These cultures were nearly covered with roots. Root
number decreased gradually at the same NAA levels when com-
bined with 5.0 or 10.0 mg/l BA. Only a few, poorly devel-
Oped roots were observed on the 10.0 mg/l BA plus 1.0, 5.0
or 10.0 mg/l NAA treatments.

Browallia gpeciosa grew very Slowly on all BA+NAA

24

 

Figure 2. Morphogenetic response of leaf sections of
Browallia viscosa to BA+NAA combinations after 6 weeks
in culture. Concentrations of BA from tOp to bottom
and NAA from left to right: 10.0, 5.0, 1.0, 0.1 and
0.01 mg/l.

 

25

e c o t

o

 

.N 023.".

(m

26

Figure 3. Morphogenetic response of leaf sections of
Browallia speggosa to BA+NAA combinations after.6
weeks in culture. Concentrations of BA from t0p to

bottom and NAA from left to right: 10.0, 5.0, 1.0,
0.1 and 0.01 mg/l.

 

& (£0 501

 

28

combinations (Figure 3). After 6 weeks, only small to
medium amounts of yellow-brown callus growth occurred even
at the highest BA+NAA concentrations. The largest amount
of callus was observed at 5.0 mg/l BA plus 10.0 mg/l NAA.
Shoots were not initiated on any of the BA+NAA combi-

nations on either Browallia Species.

 

Investigations were also carried out with leaf sec—

tions of B. Speciosa and B. viscosa on M/S with 2.5 or 0.5

 

BA plus 5.0 mg/l 2,4—D and on U/M (0.25 mg/l K plus 2.0
mg/l 2,4-D) medium. Callus was initiated and grew Slowly

on leaf eXplants of B. gpeciosa on each of these media.

 

However, callus removed from the original eXplants and sub-
cultured on the same medium increased in growth rate. The
callus produced on each of these media was whitish-yellow
and friable. Medium quantities of callus growth occurred
on 2.5 mg/l BA plus 5.0 mg/l 2,4-D, with large amounts on
0.5 mg/l BA plus 5.0 mg/l 2,4-D and on U/M.

Slow callus growth occurred when B. viscosa was cul-
tured on the Same medium. The callus which developed was
compact, whitish-yellow and no Shoot or root organization

was observed.

Shoot growth:

Shoots that arose on callus derived from both 0.1 and
1.0 mg/l 2,4-D and subcultured on 1.0 or 5.0 mg/l 2 ip were

easily rooted on.M/S medium without growth regulators, on

29

M/S+IAA at 0.01, 0.1, 1.0, 5.0 and 10.0 mg/l, or in an
artificial planting medium. Some adventitious roots also
occurred on Shoots after their elongation on Shoot regen-
eration media. Terminal Shoot cuttings placed in a moist,
artificial planting medium of peat moss and vermiculite at
a 1:1 ratio and held at 100% relative humidity rooted in
4-7 days. Roots also deve10ped within 7 days on M/S basal
medium and on I‘.T/S+IAA (Figure 4). The number and length
of roots decreased as the IAA concentration increased.
Well developed, slightly branched roots were induced on
shoots cultured on 0.01 mg/l IAA. At this IAA level, 3-5
roots, 5-38 mm in length were produced on each explant.
Shoots placed on.M/S medium deve10ped roots very Similar
to those occurring at 0.01 mg/l IAA.

Shoots rooted in the artificial planting medium were
readily acclimated to greenhouse conditions. The plants
grew vigorously and within two weeks were transferred to
10 cm pots using a sterilized 1:1:1, soilzsandzpeat moss

planting medium.
_gytology and morphology 3f regenerated plant :

Flowering Browallia viscosa plants originating from

 

callus cultured on 2 ip were evaluated visually for mor—
phological traits, cytologically and by pollen viability
studies. All regenerated plants appeared normal when first

placed in the greenhouse, but later, abnormal vegetative

30

Figure 4. Root develOpment on regenerated Shoots of
Browallia viscosa cultured on.M/S basal medium plus
IAA after 1 week'in culture. Concentrations of IAA
from left to right: 0.01, 0.1, 1.0, 5.0 and 10.0
mg/l.

 

 

32

growth was observed on some of them. One plant was one
half normal size and covered with crinkled blue flowers,
0.94 cm in diameter. Only 50% of pollen collected from
this plant was viable. This plant, subsequently did not
produce seed upon self—pollination. Unfortunately, the
chromosome number of this plant was not determined.

Several other plants also varied morphologically in
contrast to the parent B, viscosa. The leaves of these
plants were twice as large as those found on parent plants,
had more hairs on the leaf surface, and had heavier stems.
Flowers which deve10ped on these plants were of normal size
and exhibited 90% pollen viability, but seed set was re—
duced more than 50%. These plants were found to be tetra-
ploids (2n=4x=44) with 44 chromosomes.‘

Most regenerated plants upon visual evaluation matched
the parent for major morphological traits. Eighty percent
pollen viability was observed for these plants. Seed from
some of them was germinated and the seedling pOpulations
were likewise normal.

The chromosome number of each Browallia Species as

 

well as some of the regenerated plants was determined.
Browallia §peciosa and B. viscosa are diploids with chro—
mosome numbers of 44 and 22 respectively as reported by
Darlington and Wylie (1955). Most of the regenerated
plants were diploids but several tetraploids were found.
Pollen viability studies were conducted on B. S e-

ciosa, B. viscosa and on the regenerated plants. Pollen

33

from flowers of the former Species was found to be 80%
viable while 94% viability was observed with B. viscosa.

Pollen viability of regenerated plants ranged from 50-90%.

DISCUSSION

Although B. Speciosa and B. viscosa belong to the
same genus only Slight similarities existed in their re-
sponse 22 vitro. Leaf sections and callus of B. viscosa
cultured on.M/S plus auxins, cytokinins or their combina-
tions resulted in definitive morphogenetic reSponses while
eXplants Of.§' Speciosa cultured under identical conditions
died or in only a few instances initiated callus growth.
These differenced in $2 vitro morphogenetic response bet-
ween B. Speciosa and B. viscosa were not entirely unex-
pected Since there are obvious differences in growth rate,
growth habit and flower size between the two Species.
There are at least three possible explanations for the ob—
served 2p vitro differences: 1) a cultivated vs a wild
Species, 2) taxonomic differences and 3) cytological dif-

ferences.
Cultivated XE wild species:

Browallia gpeciosa is a Species that has been adapted
by breeding efforts for ornamental cultivation while B.
viscosa is a wild type. It is quite probable that Selec-

tion for flower color and Size, profuse blooming and growth

34

35
habit has indirectly modified the ability of B, Speciosa
to respond 1E 31333.
Similar contrasting LE 33333 morphological reSponseS

have been observed with other Solanaceous Species. Obser-

 

vations taken from a study of inbred Petunia Species as
well as Petunia hybrida 0v Comanche (Power et. al., 1976)
indicated that all of the inbred Species regenerated much,
easier than the cultivar Comanche. Tal et. a1. (1977)
compared morphogenetic potentials between wild and culti—
vated Species of tomato. They found that the wild Species
exhibited a higher morphogenetic potential than the cul-
tivated ones. In addition, it was noted that the morpho-
genetic responses were dependent on the auxin or cytokinin
employed. This dependency on a specific hormone has been
shown to be due to genetic control (Izhar and Pover, 1976)
and has a high degree of heritability. In studies with
several inbred Petunia Species they observed that certain
lines required 2,4-D and others NAA for growth in culture.
Hybrids between these lines were Shown to be capable of
growth on a wider range of hormones than the parents.
Frearson et. a1. (1973) also concluded that the inability
of certain Petunia hybrids varieties to regenerate when
plated on identical medium was due to genetic differences
between these varieties. In a comparison between two lines
of Lycopersicon esculentum (Ohki et. al., 1978) it was
observed that one line 'Porphyre' had a higher morphogenet—

ic capacity than the other line 'Apedice'. Reciprocal

36

crosses were made between these two parents and the hybrids
were also screened for morhpogenetic potential. They con—
cluded that the ig.3;££g response of these hybrids was ge—
netically controlled.

These findings suggest that the differential 32.23332
response between B. viscosa and B. Speciosa may be due to

genetic variation possibly as a result of breeding efforts.

Taxonomic differences:

 

It has been suggested (Sink and Power, 1977) that
variations in reSponse between several Petunia Species
were a reflection of taxonomic differences among these spe-
cies. And, upon visual comparison opr. Speciosa and B.
viscosa it became quickly apparent that taxonomic differ-

ences occur between these two Species. Browallia §peciosa

 

has a dense growth habit with large blue or white flowers
while B. viscosa has an Open growth habit and small blue
flowers with a yellow eye. These differences in turn re-
flect variable environmental conditions under which the
plants have adapted in their native habitat. And, since
in these studies both Browallia Species were grown and
cultured under identical conditions the observed differ-
ences between B. Speciosa and B. viscosa must be due to
their genotypes. From this study the conclusion may be
drawn that the differences in lg Xgigg morphogenetic re-

Sponses between B. Speciosa and B. viscosa are due to

 

37
genetic variation. Taking these arguments a step further,
it can be suggested that the observed hormonal Specificity
of these two Species must also be genetically controlled.
In 1976, Power et. a1. hypothesized that the ability

of certain Petunia Species to regenerate was correlated
with their taxanomic relationships (differences). If this
hypothesis is accepted then the large differences in mor-

phogenetic reSponseS between.B, §peciosa and B. viscosa

 

would indicate that they are not closely related taxonom-
ically. This would explain why B. viscosa has the ability
to regenerate under the culture conditions used herein and

why B, Speciosa did not.

 

Cytological differences:

 

Although both Browallia Species have a basic chromo—

 

some number of‘g = 11 and are diploid, B. gpeciosa has been
shown to have twice as many chromosomes (44) as E. viscosa
(22) (Darlington and Wylie, 1955). This difference in the
number of chromosomes also could be reSponSible for the
different reSponse of B. Speciosa and B. viscosa in cul-
ture. It is obvious that difference in reSponse between
closely related Species with a different basic chromosome

number such as between Petunia parviflora (g = 9) and B.

 

axillaris, g. violaceae and g. hybrida (all 35 = 7) (Sink
and Power, 1977) is at least partially due to the different

base chromosome number, but this could also be true between

38
Species where the basic chromosome number is the same but
the diploid chromosome number is different. This seems to
be a good eXplanation for the differences in in vitro
response between B, Speciosa and B, viscosa.

While these three eXplanations have been presented as
being somewhat distinct from each other this is not necces—
sarily the case. The three suggestions are interrelated
with the central concept being that the differences in
morphogenetic response between B. Speciosa and B. viscosa
are due to genetic variation. The cause of the genetic
variation is of primary concern. From these suggestions
also comes the conclusion that the hormone requirements

for growth 01 B. peciosa and B. viscosa in vitro are pro-

 

bably genetically controlled.

with these conclusions in mind, it becomes evident
that a discussion of the hormonal Specificity for the
growth or non-growth of B, viscosa and B. Speciosa in cul—

ture is relevant.

Auxin specificity:

 

There were few similarities in the reSponse of leaf
sections of B, viscosa and B. Speciosa when plated on.M/S
medium containing auxins. Leaf sections of B, viscosa
cultured on.M/S plus IAA, IBA or NAA deve10ped extensive
root systems. In each case, numerous fine white roots

were observed at each concentration of auxin except with

 

39

NAA at 5.0 and 10.0 mg/l. And, in this situation it is
possible that these levels may have been too high for root
initiation. Other researchers have observed similar ef—
fects with different Solanaceous Species. Gupta et. a1.
(1966) reported that IAA stimulated root develOpment di-
rectly on leaves of Nicotiana tabacum. Root initiation has
also been observed on leaf eXplants of Petunia inflata and
‘2. hybrids when supplemented with IAA or NAA (Rao et. al.,
1973 a, b).

Contrasting results were obtained when 2,4—D was used
as the auxin. Loose, friable callus deve10ped on leaf
pieces cultured on 2,4-D at 0.1 or 1.0 mg/l. Likewise,
2,4-D has been Shown to cause prolific cell growth with
Petunia (Rao et. al., 1973 a, b), Datura (Engvild, 1973)
and Solanum (Rao and Narayaswami, 1968).

Very different results were obtained when leaf sec-
tions of B._§peciosa were cultured on M/S plus IAA, IBA,
NAA or 2,4-D. Within four weeks after being placed into
culture all of the eXplants had died.

These very contrasting responses between B. viscosa
and B. Speciosa are a reflection of the genetic differences
between them. Browallia viscosa appears to have simple
genetic requirements for root initiation Since the results
are very similar with IAA, IBA or NAA. However the re-
quirements for callus induction are more Specific Since
2,4-D is required for the initiation and growth of loose

friable callus. Browallia gpeciosa represents a more

40

complex system of genetic control than B. viscosa Since it
did not respond to the incorporation of individual auxins

in vitro.

Cytokinin Specificity:

 

Contrasting morphogenetic responses were also evident

between explants of B, viscosa and B, Speciosa when cul-

 

tured on.M/S medium containing cytokinins. Browallia

 

viscosa exhibited a very Specific cytokinin requirement.
Approximately 85% of leaf sections and slightly over 50%
of callus cultures deve10ped shoots when placed on M/S
basal medium containing 2 ip. In comparison, less than
10% of SXplants cultured on K or BA initiated Shoots. The
largest quantities of callus were also observed on cultures
containing 2 ip. Ohki et. a1. (1978) reported a similar
cytokinin Specificity withqucgperSicon esculentum when

2 ip was compared with K and BA. With tomato however, the
auxin, IAA in combination with 2 ip was required to obtain
Optimum shoot proliferation.

Power and Berry (1979) reported infrequent Shoot ini-
tiation when callus derived from leaf sections of B, XLE’
'gggg was subcultured on M/S plus a BA+NAA combination or
on M/S plus Z. However, under the cultural and environ-
mental conditions used in this research, no Shoots were ob—
served on the same media formulations.

The reSponse of explants of B.§peciosa to cytokinin

 

41
treatment was quite different from that of B, viscosa and
represents a more complex hormonal requirement. Leaf and
callus eXplantS of this species were either dead or dying
within 4 or 12 weeks respectively after being subcultured
to 1.1/3 plus K, BA or 2 ip.

AS with the auxins, it is clear from the cytokinin
results that there is a great deal of genetic variation
between B. viscosa and B. Speciosa. This is represented
by the very Specific cytokinin requirement of B. viscosa
and the inability of B, Speciosa to reSpond to any cyto—

kinin treatment.

_§ytokinin/auxin Specificity:

Cytokinin/auxin combinations stimulated morphogenetic

responses with both Browallia Species that were not other-

 

wise induced. Also, each Species exhibited preferences
towards particular cytokinin/auxin combinations. Leaf
sections of B. viscosa initiated varying amounts of callus
and roots on BA+NAA combinations depending on the cyto—
kinin/auxin concentrations. In comparison, the growth rate
of B. Speciosa was much Slower than that of B, viscosa on
the same BA+NAA combinations.

Browallia §peciosa did however surpass B, viscosa in
.gg.1;££2 response with reSpect to the amount and quality
of callus growth on M/S with BA plus 2,4—D or on U/M med-
ium (0.25 mg/l K + 2.0 mg/l 2,4-D).

42

Browallia viscosa thus shows a preference for BA plus
NAA while BA or K plus 2,4-D are required for the growth of
'B.§peciosa'$g.y;££g. Similar preferential morphogenetic
responses have been observed (Izhar and Power, 1977) be—
tween several inbred Petunia Species. Following lE.XlE£9
studies with F1 hybrids between the Petunia Species with
the same and different preferences they suggested that the
Specific hormonal requirements were genetically controlled.
Their finding again indicate the genetic variation between
B. viscosa and B. Speciosa.

Sustained callus growth was also observed when callus

of B. Speciosa from a BA+2,4-D combination was subcultured

 

to H/S plus Z at 1.0 mg/l. Shoot regeneration, however,
was not observed on leaf or callus eXplantS of B, Speciosa
in these investigations.

It is interesting to note that leaf sections of B,
gpeciosa died when eXposed to individual cytokinin or auxin
levels but remained alive when they were combined in the
basal medium. Even under these conditions, only callus
growth could be induced. These findings lend support to
the hypothesis that the morphogenetic responses of B, EEE’
gigga are under a rather complex system of genetic control.
This suggests the need for changes in the eXperimental or
environmental parameters which must take place before the

regeneration of this Species can be realized.

 

43
Rooting:

While growing B. viscosa seedlings in the greenhouse
it was noted that rooting occurred when the stems of the
plants came in contact with the soil. Cuttings also rooted
easily when placed in a mist pr0pagation bench. Thus, it
was not surprising that Shoots which deve10ped in culture
rooted readily in an artificial planting medium or upon
subculture to M/S basal without growth regulators or M/S
plus IAA at 0.01 mg/l. Kartha et. a1. (1977) observed
similar results with Shoots of Bchpersicon esculentum cv
Starfire. They suggested that the ease of rooting was due
to the presence of high levels of endogenous auxins in the
tomato stems. If B. viscosa also has a high endogenous
auxin content this may account for the ease of root for-

mation.

_gytology—morphology g: regenerated plants:

The majority of plants regenerated from callus were
normal with reSpect to cytological and morphological
traits. A few were not normal in appearance, but this can
be expected in plants derived from callus cultures (Mura—

shige and Nakano, 1966 and 1967; Ohki et. a1. 1978).

44

Conclusions:

 

It has been suggested (Frearson, 1973 and Kartha et.
al., 1976) that the reSponse of leaves of a Species to
certain ig'zligg cultural conditions can be used as an in—
dicator of the ease or difficulty of using that Species
in prJtOplast studies. Thus, the results gained here with

‘B. viscosa and B. Speciosa are valuable in considering

 

them for further somatic cell studies. Both Species merit
further consideration although in different areas.
Browallia viscosa has already been regenerated from proto—
plastS (Power and Berry, 1979) and is a candidate for
further 32.23332 genetic manipulations. The fact that B,
Agpeciosa does not reSpond in culture may make it amenable
as part of a selection system for prot0plast fusion

studies.

SUMMARY

Three hypothesis have been suggested as possible ex—
planations for differences in.$2 vitro morphogenetic re—
Sponses between B. viscosa and B. Speciosa: l) cultivated
vs wild Species, 2) taxonomic differences and 3) cytologi-
cal differences. Upon cosideration of these hypothesis, it
has been concluded that the morphogenetic differences are
due to genetic variation between the two Species either
as a result of one of these suggestions or more likely due

to a combination of them.

Browallia viscosa:

 

Callus, Shoots and roots were initiated on leaf and
callus eXplants of B. viscosa cultured $3.33339. Only the
cytokinin 2 ip was effective in initiating Shoot develop-
ment. Shoots of B, viscosa rooted easily when placed on
M/S medium alone or with low levels of auxin or on an ar-
tificial planting medium. Growth regulators were not re-
quired for a high percentage rooting of shoots. Plants
could be regenerated from callus cultures and were suc-
cessfully grown to flowering in the greenhouse. Most of

the regenerated plants were observed to be identical to the

45

46

parent, B. viscosa by following visual, cytological and

pollen viability evaluation.

Browallia Speciosa:

 

Browallia Speciosa did not respond in culture. Callus

 

deve10pment occurred only when leaf sections were cultured
on cytokinin/auxin combinations. Prolific callus deve10p-
ment occurred on M/S basal medium with 0.5 or 2.5 mg/l

BA plus 5.0 mg/l 2,4-D and on U/M. No Shoots deve10ped

on leaf eXplants or callus cultures of B, Speciosa 12

vitro.

BIBLIOGRAPHY

BIBLIOGRAPHY

Bailey, L. H. 1976. Hortus Third. MacMillian Publishing
Co. Inc., New York. I29O pp.

Darlington, 0. D. and L. F. La Cour. 6th ed. 1975. The
Handling 2: Chromosomes. John Wiley and Sons, New
York.

 

 

Darlington, 0. D. and A. P. Wylie. 2nd ed. 1955. Chromo-
some Atlas of Flowering Plants. George Allen an
Unwin L.T.D., London.

Engvild, D. C. 1973. Shoot differentiation in callus
cultures of Datura innoxia. Physiol. Plant. 28:

 

Frearson, E. M., J. B. Power and E. C. Cocking. 1973.
The isolation, culture and regeneration of Petunia
leaf prot0plasts. Developmental Biology 33: I35-

137 o

 

Guha, S. and S. C. Maheshwari. 1964. In vitro production
of embryos from anthers of DaturaI—Nature 204: 497.

Gupta, G. R. 3., S. Guha and S. C. Maheshwari. 1966.
Differentiation of buds from leaves of Nicotiana
tabacum Linn. in sterile culture. Phytomorphology

I6: 175-182.

Halperin, W. 1966. Alternative morphogenetic events in
cell suspensions. Amer.'g. Bot. 53: 443-453.

 

Halperin, W. and D. F. Wetherell. 1964. Adventive embry-
ony in tissue cultures of the wild carrot, Daucus
carota. Amer. g, Bot. 51: 274-283.

Handro, W., P. S. Rao and H. Harada. 1972. Controle
hormonal de la formation de cals, bourgeons, racines
et embryos sur des explantats de feuilles et de tiges
dc Petunia cultivar in vitro. 0. B, Acad. B33,

(Paris) 275(13): 2861228637 "

47

 

48

Hughes, H., S. Lam and J. Janick. 1973. BB vitro culture
of Salpiglossis sinuata L. Hortscience 8: 335-336.

 

Izhar, S. and J. B. Power. 1977. Genetical studies with
Petunia leaf prot0plasts. 1. Genetic variation to
Specific growth hormones and possible genetic control
on stages of prot0plast deve10pment in culture. Plant
B2B, Letters 8: 375-383.

Johansen, D. A. lst ed. 1940. Plant Microtechnique.
McGraw-Hill Book Co. Inc. ‘New York

Kartha, K. K., S. Champoux, O. L. Gamborg and K. Pahl.
1977. BB vitro pr0pagation of tomato by Shoot apical
meristem cuIture. J. Amer. Soc. Hort. Sci. 102:

346-349. — — — —_ —

Kartha, K. K., O. L. Gamborg, J. P. Shyluk and F. Constabel.
1976. Morphogenetic investigations on in vitro leaf
culture of tomato (Ly00persicon esculentfim.M1II. 0v.
Starfire) and high frequency plant regeneration. B,
Pflanzenphysiol. 77: 292-301.

Lee, C. W., R. M. Skirvin, A. I. Soltero and J. Janick.
1977. Tissue culture of Sal i lossis sinuata L. from
leaf discs. HortScience. IE: 547-549.

Linser, H. and K. H. Neumann. 1968. Untersuchungen fiber
Beziehungen zwishen Zellteilung und Morphogenese bei
Bewebekulturen von Daucus carota. Physiol. Plant.
21: 487-499.

Murashige, T. and R. Nakano. 1966. Tissue culture as a
potential tool in obtaining polyploidy plants. .1-
Heredity 57: 114-118.

 

Murashige, T. and R. Nakano. 1967. Chromosome complement
as a determinant of the morphogenetic potential of
tobacco cells. Amer. g. Bot. 54: 963-970.

Murashige, T. and F. Skoog. 1962. A rivised medium for
rapid growth and bioassays with tobacco tissue cul-
tures. Physiol. Plant. 15: 473-497.

Norton, J. P. and W. G. B011. 1954. Callus and Shoot
formation from tomato roots lg vitro. Science 119:
220-221.

Ohki, S., C. Bigot and J. Mousseau. 1978. Analysis of
shoot-forming capacity 22 vitro in two lines of tomato
(Ly00persicon esculentum.MiII.) and their hybrids.
Plant 2E9 Oell Physiol. 19: 27-42.

 

 

49

Padmanabhan, V., E. F. Paddock and W. R. Sharp. 1974.

Plantlet formation from L 00 ersicon esculentum leaf
callus. Can. B. Bot. 52: I429—l432.

Power, J. B. and S. F. Berry. 1979. Plant regeneration
from prot0plasts of Browallia viscosa. B, Planzen-

physiol. 94: 469—471.

Power, J. B., E. M. Frearson, D. George, P. K. Evans, S. F.
Berry, C. Hayward and E. C. Cocking. 1976. The
isolation, culture and regeneration of leaf prot0plasts
in the genus Petunia. Plant B3B. Letters 7: 51-55.

Rao, P. S., W. Handro and H. Harada. 1973a. Hormonal
control of differentiation of Shoots, roots and
embryos in leaf and stem cultures of Petunia inflata

and Petunia hybrida. Physiol. Plant. 28: 458-463.

Rao, P. S., W. Handro and H. Harada. 1973b. Bud formation
and embryo differentiation in in vitro cultures of
Petunia. B, Pflanzenphysiol. _69: 87-90.

Rao, P. S., and S. Narayanswami. 1968. Induced morpho-
genesis in tissue cultures of Solanum xanthocarpum.

Planta 81: 372-375.

Reinert, J. 1959. Uber Die Kontrolle Der Morphogenese und
die Induction Von Adeventivembryonen an Gewebekulturen
AuS Karotten. Planta 53: 318-333.

Skoog, F. and C. Tsui. 1948. Chemical control of growth
and bud formation in tobacco stem segments and callus
cultured $2 vitro. Amer. B, Bot. 35: 782-787.

Skoog, F. and W. Miller. 1957. Chemical regulation of
growth and organ formation in plant tissues cultured
$2 vitro. Sy p. Soc. E p. Biol. 11: 118-131.

Sink, K. C. and J. B. Power. 1977. The isolation, culture
and regeneration of leaf prot0plasts of Petunia ar-
viflora Juss. Plant Sci. Letters 10: 335-340.

Swamy, R. D. and E. K. Chacko. 1973. Induction of plant-
lets and callus from anthers of Petunia axillaris
(Lam.) B.S.P. cultured $2 vitro. Hort. Res. 13:
41-44 0

Tal, M., K. Dehan and H. Heiken. 1977. Morphogenetic
potential of cultured leaf sections of cultivated
and wild Species of tomato. Ann. Bot. 41: 937-941.

50

Uchimiya, H. and T. Murashige. 1974. Evaluation of para-
meters in the isolation of viable prot0plasts from
cultured tobacco cells. Plant Bysiol. 54: 936—944.

Walkey, D. G. A. and J. M. G. Woolfitt. 1968. Clonal
multiplication of Nicotiana rustica L. from shoot
meristems in culture. Nature 220: 1346-1347.

White, P. R. 1934. Potentially unlimited growth of
excised tomato root tips in a liquid medium. Plant

Physiol. 9: 585-600.

Zenkteler, M. 1972. BB vitro formation of plants from
leaves of Several SpeCles of the Solanaceae family.
Biochem. Physiol. Pflanzen. 163: 509-512.