Basella alba L. is a tropical vine used as a vegetable in some Asian and African countries. It has potential as a nontraditional crop for small family farms. A short day plant, it blooms during the fall, provided the temperatures are mild. In the southeastern U.S., the short days of fall are associated with subfreezing temperatures, and plants are killed before blooming. Attempts were made to regenerate the plant using tissue culture techniques. Several trials were conducted with different media, hormones, and explants. It was found that nodal segments on Gamborg medium regenerated shoots. Interaction studies of auxins and cytokinins indicated that its endogeneous auxin content might be high because callus proliferated in almost all treatments and roots initiated even when the medium was not supplemented with an auxin

Edney, Norris Allen

Rizvi, Muhammad A.

Rizvi, Narjis F.

English

NASA Technical Reports Server

N91-28069
A
IN VITRO REGENERATION OF BASELLA ALBA L.
Norris Allen Edney, Muhammad A. Rizvi
and Narjis F. Rizvi
Department of Biological Sciences
Alcorn State University
Lorman, Mississippi
/
ABSTRACT
I_sy,]la..alt_i. is a tropical vine used as a vegetable in some Asian and African countries. It has
potential as a non-traditional crop for small family farms. A short day plant, it blooms during fall,
provided the temperatures are mild. In the southeastern United States, the short days of fall are
associated with sub-freezing temperatures, and plants are killed before blooming. In this study attempts
were made to regenerate the plant using tissue culture techniques. Several trials were conducted with
different media, hormones and explants. It was found that nodal segments on Gamborg medium
regenerated shoots. Interaction studies of auxins and cytokinins indicated that its endogenous auxin
content might be high because callus proliferated in almost all treatments and roots initiated even when
the medium was not supplemented with an auxin.
33
https://ntrs.nasa.gov/search.jsp?R=19910018755 2020-03-19T16:44:50+00:00Z
IN VITRO REGENERATION OF BASELLA ALBA L.
Introduction
In recent years an increasing interest has been expressed in non-traditional crops that show
promise for small American family farms (Tiwari et. al., 1988). _ could be a promising new
vegetable crop for small farms because it is pest and disease resistant, it has a high nutritive value, and
its yield is 8 times higher than regular spinach (Yamaguchi, 1983).
]_c, lla..alka_, is a tropical vine (Bailey, 1951). It is used as a vegetable in some countries of
Southeast Asia CYamaguchi, 1983 and Tiwari et. al., 1988). Basella leaves and stems taste like spinach;
therefore, it is known as "Malabar Spinach" in English (Yamaguchi, 1983). In India and Bangladesh it
is known as "Poi Sag" (Tiwari et. al., 1988). In China it is called "Lok Kwa" (Yamaguchi, 1983).
B_gll_di2al. blooms during the short days of fall provided the temperatures are mild at nights.
In Mississippi and all other parts of the southeastern United States short days are associated with
sub-freezing temperatures at night. Therefore, ]_al_dlagtll_tL vines die before flowering.
Plant tissue culture techniques have been valuable in alleviating such problems through vegetative
propagation of plants (Evans et. al. 1983). Plant regeneration _ is the key to practical application
for most purposes using tissue culture techniques (Tomes, 1985).
Regeneration of any member of ])._gJla.alb.a_. has not been reported with the use of tissue culture
techniques. Therefore, the purpose of this study was to investigate regeneration of ]_Le]likall21LL. in
vitro.
Materials and Methods
This study was conducted in three phases. The objective of phase I was to compare the perfor-
mance of the three most used plant tissue culture media: Murashige & Skoog (MS), Anderson (AND)
and Gamborg B5 (GB5) on callus proliferation, and root and shoot initiation on five explants: petiole,
stem, meristem, leaf and node. Explant used were 0.5 cm 3 in size or segments of stem, leaf, petiole,
meristem and node. All of the explants were put in Murashige and Skoog (MS), Anderson (AND) and
Gamborg B5 (GB5) media after surface sterilization. Explants were sterilized as described by Thorpe
(1981). MS medium had 100, 0.5, 0.5, 0.1, 2.0 and 80.0 mg/l of myo-inositol, nicotinic acid, pyrodoxine
HCI, thymine HCI, glycine and adenine sulfate (Hartman and Kester, 1983). AND medium was
supplemented with 4 mg/l thiamine HCI, 100 mg/l myo-inositol and 80 mg/l adenine sulfate (Hartman
and Kester, 1983). GB5 medium had 10.0, 1.0, 100.0 and 80.0 mg/l of thiamine HCI, nicotinic acid,
pyridoxine HCI, myo-inositol and adenine sulfate respectively in addition to basal inorganic salts
(Hartman and Kester, 1983). Growth regulators for callus proliferation, shoot and root initiation trials
were 0.1, 0.2, 0.4, 0.8 and i.6 mg/l of napthaleneacetic acid (NAA) as auxin and 0.1, 0.2, 0.4, 0.8 and 1.6
mg/l of benzyl adenine (BA) as cytokinin. In every trial, one set without growth regulators was used as
control. The pH of the media were adjusted to 5.7 before autoclaving. All three culture media had 30
g/l sucrose and 7 g/1 agar (Thorpe, 1983). Media were autoclaved at 121°C for 20 minutes. The culture
environment was maintained at 25°C and illuminated with cool white fluorescent lamps at 40 u
34
Einsteins/m2/seefor 16hoursperday. After 3 weeks the cultures were evaluated by determining the
size of calluses and total number of shoots greater than 5 mm in length produced per explant.
The objective of the phase II experiments was to evaluate the interactions of napthalenenacetic
acid (NAA) and benzyl adenenine (BA) on callus proliferation and initiation of roots and shoots from
explants selected earlier in phase I. It was 6 x 6 diallel experiment (Sondahl et. al., 1981). Dilution of
NAA and BA were 0, 0.1, 0.2, 0.4, 0.8 and 1.6 ppm. Each treatment had 5 replications.
Mature vines were used as source of explants. The explants used were 3 - 5 m m segments of nodes.
The explants were surface sterilized with 20% chlorox for 5 minutes and washed with distilled water 5
times. (Thorpe, 1981). Standard Gamborg B-5 medium was used as a source of nutrition for explant
growth. It was supplemented with 3% sugar and 0.8% agar. After addition of NAA and BA, the pH of
the medium was adjusted to 5.7 - 5.8 by NaOH and/or HCI solution. The medium was sterilized and
autoclaved at 121°C at 15 Ibs. PSI for 15 minutes.
Results and Discussion
Five explants: leaf, stem, meristem, petiole and nodal segments, and three media: MS, AND and
GB-5 were used in phase I experiments. It was found that all explants initiated calluses but shoot
initiation was restricted to a few combinations. Leaf explants on all three media showed signs of cell
growth and cell elongation but did not produce a well defined callus. Stem, meristem and petiole
initiated calluses on all three media are under investigation. Nodes produced calluses and proliferated
shoots on GB5 and AND media but not on MS medium. In primary cultures shootinitiation occured
only with the use of GB5 and AND media. Leaf, stem, meristem and petiole when used on shooting
medium initiated calluses. The failure of leaf, stem, meristem and petiole explants to produce shoots
on any of the media was not unprecedented. Endogenous hormones play a very significant role in the
outcome of exogenous hormones on shoot proliferation (Band and Lineveger, 1986). Because of poor
shooting responses on MS and AND media, further studies were made on GB5 medium only.
In the interaction trials it was found that the effective dilutions of NAA and BA for initiation of
shoots ranged in between 0 - 1.6 ppm (Table 1).
35
Table 1:
Effect of NAA and BA in GB5 medium on shoot regeneration
from nodes of _ after two weeks of inoculation.
BA NAA Percent of culture
rag/1 mg/1 with shoots
0 - 0.1 0 - 0.1 9.3
0.8 - 1.6 0 - 0.1 89.4
0.2-0.8 0.2-0.8 5.7
In the second experiment of interaction of NAA and BA it was found that calluses were observed
in all of the treatments (Table 2).
Table 2:
Interaction of NAA and BA on callus formation.
BA in ppm
0
NAA in ppm
0 0.1 0.8 1.6
0.1 1.2" 0.6 2.0 2.0
0.2 1.7 0.8 2.0 2.0
0.4 1.6 1.2 2.0 2.0
0.8 1.6 1.2 2.0 2.0
1.6 1.6 1.2 2.0 2.0
*xexplantsize
0.2 0.4
1.3 2.0
1.3 2.0
1.8 2.0
1.8 2.0
1.8 2.0
36
Shoots initiated only when NAA was in the range Of 0 to 0.1 ppm. Higher concentrations of NAA
inhibited shoot formation (Table 3).
Table 3:
Interaction of NAA and BA on shoot formation.
BA in ppm
0.1
0.2
0.4
0.8
1.6
0.1
2 •
6
NAA in ppm
0.2 0.4
1
0.8 1.6
* number of shoots/treatment
NAA favored root formation. Absence of BA was not critical for root formation. In some
treatments, root initiation was found even in high concentrations of BA, but such incidence was rare
(Table 4).
In phase II it was found that the favorable ratios for rooting and shooting were 0.1 - 0.2 ppm of
BA/0.8 - 1.6 ppm of NAA, and 0.8 - 1.6 ppm of BA/0.1 - 0.2 ppm of NAA respectively. Media with the
same ratios were used in phase III of this experiment. It was found that calluses did not respond to these
treatments. Further studies are suggested to be conducted with the use of other hormones.
37
Table4:
Interactionof NAA andBA on root formation.
BA in ppm
0.1
0.2
0.4
0.8
1.6
NAb. in ppm
0.1
2 •
0.2
2
0.4
6
6
0.8
8
1.6
8
* number of roots/treatment
Literature Cited
1. Bailey, L. H. (1951). Manual of Cultivated Plants. Macmillan Co., New York. p. 367.
2. Brand, Mark H. and R. Daniel Linebeger (1986). In Vitro Propagation of Halesia Carolina
L. and the Influence of Explantation Timing on Initial Shoot Proliferation. Plant, Cell,
Tissue and Organ Culture: Mar. Nuj. Publisher, Netherlands.
3. Evans, D., W. Sharp, P. Ammirato and Y. Yamada (1983) Handbook of Plant Cell Culture.
Macmillan & Co., New York p. 126.
4. Hartman, Hudson T., and Dale E. Kester (1983). Plant Propagation, Prentice Hall Inc.,
Englewood Cliffs, New Jersey.
5. Thorpe, Trever A. (1981). Plant Tissue Culture. Academic Press., Orlando, FL.
6. Tiwari, S. C., P. E. Igbokwe and J. B. Collins (1988) Malabar Spinach (BAsgdla_aLL.)
MAFES Information Sheet 1321, Miss. State University, MS 29762.
38
.o
.
Tomes, Dwight T. (1985). Opportunities and Limitations of Genotypic Influence on
Establishment of Plant Regeneration from Callus and Cell Cultures of Crop Species in
Biotechnology in Plant Science. Eds: Zaitlin, Day and Hollaender. Academic Press.
London.
Sondahl, M. R., L. C Monaco, and W. R. Sharp (1981). In Vitro Method Applied to
Coffee in Plant Tissue Culture. Ed. Thorpe. Academic Press, Orlando, FL. p. 325.
Yamaguchi, M. (1983). Other Succulent Vegetables. World Vegetables: Principles,
Production and Nutritive Values. AVI Publication Co., Westport, CT., p. 267-368.
39


In vitro regeneration of Basella alba L

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