TISSUE CULTURE STUDIES OF NINE PEA CULTIVARS

PNL Volume 19 1987 RESEARCH REPORTS 93

Yenne, S. P., D. C. Thill, D. J. LeTourneau, and D. L. Auld

University of Idaho, Moscow, ID USA

A large number of plant cells can be screened for herbicide
resistance or tolerance in a relatively small space and a short
period of time using tissue culture systems. However, the proper
nutrient, vitamin, and hormone concentrations needed to induce
callus growth and cell proliferation are not known for many crop
species (3,4,5). Jacobsen et al. (5,6,7) reported that callus
formation, regeneration, and in vitro differentiation of peas
(Pisum sativum L.) in tissue culture are not only medium specific,
but also genotypic specific. The purpose of the present experi-
ment was to develop media for good callus formation and cell pro-
liferation of several pea cultivars in suspension culture.

Nine pea cultivars were used: 'Scout', 'Paloma', 'Alaska',
'Garfield', and 'Latah' (spring-types), and 'Frogel', 'Glacier',
'Melrose', and 'Common' (winter-types). Fifty seeds of each cul-
tivar were surface sterilized for 5 min in 0.5% sodium hypochlo-
rite and then were rinsed three times with sterilized, distilled
water. Seeds of each cultivar were placed in sterile petri dishes
on Whatman #1 filter paper moistened with 5 ml of sterile water.
Petri dishes were wrapped with aluminum foil and placed in a
growth chamber for 3 d at 24C. Seedlings then were surface steri-
lized for 5 min with 5% sodium hypochlorite and triple rinsed with
sterile, millipore filtered water. Using aseptic techniques, the
cotyledons, root, and shoot were removed from each seedling lea-
ving only the cotyledonary node. Three cotyledonary nodes of each
cultivar were transferred into 125 ml Erlenmeyer culture flasks
containing 25 ml of liquid medium and/or into separate culture
bottles containing 25 ml of Blaydes' medium solidified with 8 g/l
of agar (Table 1). Growth regulators were used at the following
rates: picloram (4-amino-3,5,6-trichloro-2-pyridinecarboxylie

acid) at 0.1 mg/l, 2,4-D (2,A-dichlorophenoxy acetic acid) and NAA
(naphthylacetic acid) at 1.0 mg/l, and kinetin at 0.5 mg/1. All
media were supplemented with 1.0 mg/l of nicotinic acid, 1.0 mg/l
of pyridoxine-HCl, 10.0 mg/l of thiamine-HCl, and 100 mg/l of
inositol. Liquid cultures were placed on an orbital shaker (125
rpm) and incubated at 24C in the dark. Fresh medium was added
every 15 to 20 d until cells could be inoculated onto prolifera-
tion medium. Callus cultures were incubated in the dark at 24C.

Table 1. Pea cultivars tested in different media and
combinations of growth regulators used to induce callus formation.


Blaydes' solid	B5 liquid			MS liquid
kinetin + 2,4-D	NAA	2,4-0	picloram + kinetin	picloram	picloram +• kinetin
Glacier Melrose Common Alaska Latah Frogel Scout	Alaska Latah Garfield Melrose Common Glacier	Alaska Latah Garfield Glacier Mel rose	Alaska Glacier Garfield Common Mel rose	Garfield Alaska	Latah Paloma Scout Common Alaska

PNL Volume 19 1987 Research Reports

After the cultures were established on induction media (15 to
45d depending on cultivar), subcultures were transferred to proli-
feration media. Cultures initiated on Blaydes' solid medium (1)
were subcultured to Blaydes' liquid medium that contained 0.5 mg/l
kinetin and 1.0 mg/l 2,4-D or picloram plus kinetin were subcul-
tured to B5 media, which contained kinetin at 0.5 and NAA or 2,4-D
at 1.0 mg/l. Cultures initiated on MS liquid medium with picloram
plus kinetin were transferred to MS medium that contained 1.0 mg/l
of NAA and 0.5 mg/l of kinetin. Inoculum size from Blaydes' solid
medium varied from the original explant to 100 mg of callus depen-
ding upon the cultivar. Ten ml aliquots of liquid media con-
taining from 0.2 to 1.0 ml of packed cells were used to inoculate
liquid media. Addition of casein hydrolysate at 2 g/l to proli-
feration media was evaluated for increased cell proliferation.
The use of glucose versus sucrose also was compared with the cul-
tivar Glacier in the B5 proliferation medium with 1.0 mg/l NAA and
0.5 mg/l kinetin. Visual evaluations of callus formation and/or
cell suspension formation were made every 15 d and were rated from
no formation (-) to good formation (+++).

Table 2. The effect of media on induction and
proliferation among pea cultivars.


Cultivar	Induction	Proliferation
	Blaydes' solid	Blaydes' liquid
Glacier	-*
Garfield	++	+
Latah	++	-
Scout	+++	-
Common	-
Frogel	+++	+++
Melrose	+++	+++
Alaska	-
	B5/2.4-D	B5/2.4-D+K** B5/NAA+K
Alaska	-
Latah	-
Garfield	-
Glacier	++	++ ++
Melrose	+	+ -
	B5/P*** + K	B5/2.4-D+K B5/NAA+K
Alaska	-
Common	-
Garfield	+	+ +
Glacier	+++	++ +++
Melrose
	MS/P + K	MS/NAA+K
Alaska	-
Common	++	-
Latah	-
Pa 1oma	-
Scout	+	-

* no callus growth, + poor callus growth, ++ fair callus growth,
+++ good callus growth.
**K = kinetin
***P = picloram.

PNL Volume 19 1987 RESEARCH REPORTS

None of the media and hormone combinations used in this ex-
periment induced callus in all 9 cultivars. The best callus
growth was initiated on Blaydes' solid medium for five of eight
cultivars (Table 2). However, only three of the five cultivars
proliferated in Blaydes' liquid medium. Callus was not induced on
B5 medium with NAA, but some shoots (not more than 1 per explant)
and roots were induced on Melrose and Glacier (data not presen-
ted). Callus growth was induced in the cultivars Glacier and Mel-
rose with B5 liquid and 2,4-D. Callus proliferation of Glacier
was fair when cells were transferred from B5 with 2,4-D to B5 with
kinetin and 2,4-D or NAA. B5 plus picloram and kinetin induced
good callus growth in Glacier, and when these cells were trans-
ferred, proliferation was fair on B5 with 2,4-D plus kinetin and
good on B5 with NAA plus kinetin. Glacier formed some embryoid-
like structures in the B5 medium with picloram and kinetin. MS
medium with picloram and kinetin induced some callus in Common and
Scout, but neither cultivar proliferated on MS with NAA plus kine-
tin (Table 2). Callus growth was not induced in Alaska or
Garfield on MS medium with picloram. There were no differences in
callus growth of Glacier in glucose or sucrose B5 medium (data not
presented). Two grams of casein hydrolysate/l added to the proli-
ferating media increased cell growth in all cultivars that were
successfully proliferated (data not presented).

1. Blaydes, D. F. 1966. Physiol. Plant. 19:748-753.

2. Gamborg, 0. L., T. Murashige, T. A. Thorpe, and I. K. Vasil.
1976. In Vitro 12:473-478.

3. Green, C. E., R. L. Phillips, and R. A. Kleese. 1974. Crop
Sci. 14:54-58.

4. Herlt, M., H-J. Jacobsen, A. Rasche, and H. W. Ingensiep.
1978. PNL 10:20-21.

5. Jacobsen, H-J., H. W. Ingkensiep, M. Herlt, and

M. L. H. Kaul. 1980. Plant Cell Cultures: Results and

Perspectives. F. Sala, B. Parisi, R. Cella, and

0. Cifferri (Eds.). North-Holland Biomedical Press.

6. Jacobsen, H-J. and W. Kysely. 1984. Plant Cell Tissue
Organ Culture 3:319-324.

7. Jacobsen, H-J. and A. A. Salha. 1984. PNL 16:27-28.

****