Boletim de Pesquisa 245
e Desenvolvimento
ISSN 1676 - 340
Fevereiro, 2009
Encapsulation of Cassava nodal
segments for germoplasm storage
ISSN 0102 0110
Fevereiro, 2009
Empresa Brasileira de Pesquisa Agropecuária
Embrapa Recursos Genéticos e Biotecnologia
Ministério da Agricultura, Pecuária e Abastecimento
Boletim de Pesquisa
e Desenvolvimento 245
Encapsulation of Cassava nodal
segments for germoplasm storage
L. Pedro Barrueto Cid
Andréa R. Ramos Cruz
Luiz Joaquin Castelo Branco Carvalho
Embrapa Recursos Genéticos e Biotecnologia
Brasília, DF
2009
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Figura da Capa: Bead after 30 days at 15 ◦C, seen under simple microscope.
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Sumário
Abstract ................................................................................................................. 5
Resumo .................................................................................................................. 6
Introduction............................................................................................................ 7
Materials and methods .......................................................................................... 7
Results and discussion .......................................................................................... 9
Literature .............................................................................................................. 11
Encapsulation of Cassava nodal
segments for germoplasm storage
L. Pedro Barrueto Cid 1
Andréa R. Ramos Cruz 2
Luiz Joaquin Castelo Branco Carvalho1
Abstract
Caulinary nodes (axillary buds) from in vitro plants of three cassava genotypes were: (A)
encapsulated with Ca-alginate and placed on SP medium; (B) encapsulated and placed on wet
filter paper, (C) non-encapsulated and placed on SP medium; (D) non encapsulated but, placed
on wet filter paper. All treated nodes were kept during 30 days at 15°C under dark and finally
transferred to 27°C ± 2 and 16h light for 30 days more. High percentages of plant recovers
(> 60 %) were observed in A and C treatments without signs of plantlet damage. Conversely,
plant recovers from wet filter paper, B and D, were absent. This opens up another possibility
for cassava long-term storage using in vitro culture techniques.
Additional index words: Manihot esculenta; Ca-alginate; clonal storage; and SP medium.
1
Researchers. EMBRAPA Genetic Resources and Biotechnology, C.P. 02372, CEP 70849-970, Brasília-DF, Brazil.
2
Under-graduate student
Encapsulamento de segmentos nodais de mandioca para armazenamento
de germoplasma.
Resumo
Nós caulinares de mandioca oriundos de três genótipos cultivados in vitro foram: (A)
encapsulados com alginato de cálcio e colocados em meio básico SP; (B) encapsulados e
colocados em papel filtro umedecido, (C) não encapsulados, porém, colocados em meio
básico SP; (D) não encapsulados e colocados em papel filtro. Todos os tratamentos foram
mantidos por 30 dias no escuro e 15 ◦C e logo depois, transferidos para luz, 16h, e
temperatura de 27 ± 2 ◦C. Nestas últimas condições, alta percentagem de conversão de
plantas (> 60 %) foi observada nos tratamentos A e C, inclusive, sem sinais de dano no
material clonal. Em contraste, a conversão em plantas, nos tratamentos B e D, não foi
observada. Estes resultados abrem promissoras possibilidades para o armazenamento de
germoplasma de mandioca por longo tempo, usando a cultura de tecido in vitro.
Palavras chaves: Manihot esculenta, alginato de cálcio, armazenamento e meio SP básico.
7
Introduction
Cassava (Manihot esculenta Crantz) is a very important staple food because of its starchy
root. Brazil produces 31% of the global production (MATTOS e DANTAS, 1981). Novel
mutants with potential agronomic traits have been found with high levels of free sugar, βcarotene, lutein, and lycopene content (CARVALHO et al., 2000; CARVALHO et al., 2004).
Nearly of 56% of the 97 wild species of Manihot are found in Latin America. Unfortunately
for diverse reasons some of these landraces are being lost and urgently need to be saved
(GROS et al., 2000). The maintenance of part or all of this germplasm in experimental or
nursery fields is laborious and expensive. Plant tissue culture provides a useful tool to
maintain this material with low cost under laboratory conditions (BARRUETO CID e
CARVALHO, 2008).
Synthetic seeds production by encapsulation with calcium alginate of axillary buds or somatic
embryos and low temperature storage has been reported for various agronomic crops (BAPAT
et al., 1987; REDENBAUGH, 1990; GHOSH e SEN, 1994). It has been shown that storage of
synthetic seeds can alleviate frequent subculture practice in germplasm collection
maintenance, facilitate germplasm transport in activities related to germplasm exchange
between partners (countries or laboratories), in addition to it offers easy handling material for
the scale up of elite clones (PICCIONI e STANDARDI, 1995; GANAPATHI et al., 2001; LISEK
e ORLIKOWSKA, 2004).
The present study describes a method for the encapsulation of axillary buds specifically for
cassava to accesses its potential for in vitro long-term germplasm conservation.
Materials and methods
a) Cuttings.
Cassava cuttings (Manihot esculenta Crantz) (200 mm - 300 mm) from two landraces: CAS
36.18; F 50.77 and from cultivars IAC each having several dormant axillary buds, were
collected at the experimental Embrapa/Cenargen field in Brasilia DF. Cuttings were transferred
to the greenhouse and grown in black polyethylene bags containing vermiculite (1 kg
capacity). Cuttings were kept moist under environment temperature of 23 ± 3 °C. After 30
days they started to sprout. When the shoots reached ca. 100 mm (long) by 5mm (width),
they were removed and taken to laboratory.
b) Surface sterilization and inoculation
Shoots were washed with tap water and detergent and cut into segments of 10 to mm – 15
mm each with one axillary bud (mini-cutting).
In a laminar air-flow cabinet the mini-cuttings were placed in commercial bleach (sodium
hypocloride 2.5 % (w/v) for 10 minutes and washed with distilled de-ionized water (DDW).
8
After this procedure, the mini-cuttings were transferred to a glass beaker having a sterile
solution of Benlate 500 (Dupont) and Claforan (Hoechs/Roussel) 0.1 % (w/v) respectively,
and stirred for 30 min.
The mini-cuttings were inoculated in assay tubes (22 mm x 150 mm) having 15 ml of SP
medium (BARRUETO CID e CARVALHO, 2008) and kept under standard conditions of light
and temperature (27 ± 2 °C and 16 h photoperiod with a photosynthetic photon flux of 50
µmol m-2 s-1 emitted from cool-white fluorescent tubes).
After 45-60 days, the shoots obtained from axillary buds now 10 mm-20 mm long were
removed and transferred to assay tubes containing 15 ml fresh SP medium. After 45 days
under standard conditions, each shoot now ca. 50 mm long, produced 4 or 5 nodes with one
axillary bud per node and two or three roots per shoot (ca. 20 mm-30 mm long).
c) Alginate treatment
Plantlets were withdrawn from the assay tubes and their leaves and roots were removed. The
caulinary node was cut into 5-6 mm segments each having one axillary bud per node (Fig.1
A). The segments were transferred to a 50 ml glass beaker with 3 % (w/v) sodium alginate
(500 cps, Nacalai Tesque Inc. Kyoto-Japan) and 2 % (w/v) sucrose.
Using a Pasteur pipette with tip cut off, the nodes were then placed into 80 ml of CaCl2.2H20
(1 % w/v) for the preparation of drops each having one node. The CaCl2 solutions were kept
on a magnetic stirrer (ca. 80 rpm) for 30 minutes (Fig. 1B). The beads that were formed in
the CaCl2 were sieved (plastic sieve of 50 mm of diameter) and placed into an open Petri dish
and were not rinsed, Fig. 1 C. The beads containing the nodes, Fig. 1C, were submitted to
different treatments.
Fig. 1. A: Visual aspect of caulinary nodes with axillary buds (~ 6,0 mm of length) CAS 36.18.B: encapsulated node
induction, beads, in Ca-Cl2 solution kept on a magnetic stirrer. C: beads isolated and sieved from CaCl2 solutions. D:
Visual aspect under simple microscope of a bead (~ 7 mm of length) after 30 days at 15 ◦C. E-F: axillary bud
sprouting and growing from bead respectively (A treatment).
9
The following group treatment were made: A: encapsulated nodes were placed in a Petri dish,
100 mm x 10 mm with 25 ml SP medium. B: encapsulated nodes were inoculated into a Petri
dish containing two filter paper layers and 5 ml of DDW. C: nodes were not encapsulated and
placed into a Petri dish with 25 ml SP medium. D: nodes were not encapsulated and placed
into Petri dish with two filter paper layers and 5 ml DDW. E (control) similar to A, but kept at
standard conditions of light and temperature without prior exposure to 15 °C.
All sterile-disposable Petri dishes with nodes: A, B, C, D and E were sealed with plastic film.
All except E were then kept in a dark incubator for 30 days at 15°. Claforan was sterilized
using a Millipore filter (0.2µm pore). The material inside the laminar flow hood (glassware,
forceps, alginate and CaCl2 solutions, SP medium, etc.) was autoclaved at 120 °C for 20
minutes.
The experimental design consisted of 12 nodes per Petri dish. All treatments with a total of
36 nodes were replicated three times.
Results and discussion
Under standard conditions of temperature and light, nodes of the different clones inoculated
into SP medium showed high percentages of conversion into plants (Table 1: A, C; Fig.1E).
No oxidative browning was observed. After 30 days the plantlets in tubes were 20-30 mm
high and produced several roots (Fig. 1 F). For all nodes inoculated on filter paper plus DDW,
encapsulated or not ( Table 1: B-D), the plant conversions after 30 days was zero. No stem or
roots were formed because the axillary bud did not sprout or grow. Other authors reported
similar results for plant conversions in nutritive medium and filter paper (GANAPATHI et al.,
2001).
Table1. Effect of different treatments on cassava axillary buds “germination” under standard
conditions of light and temperature after treatment under 15 ◦C and darkness (except E).
Genotype
A
B
C
D
E
CAS 36.18
86± 5
0
79± 6
0
83± 23
IAC
79± 6
0
67± 0,0
0
79± 6
F 50.77
70± 18
0
66± 12
0
62± 6
Average frequency (%) ± standart deviation
The lack of plant conversion from axillary buds into plants (B-D treatments), was not
attributed to the temperature at 15 °C. The alternative treatments (A-C) was kept at the same
temperature, but plants developed from the calcium alginate beads.
At low temperature, axillary buds remained dormant during the 30 days, Fig. D. No signs of
rooting or shooting were seen emerging from beads. Cassava being a tropical plant, we do
10
not know why the 15 °C was sufficient to stop axillary bud growth during the short storage.
This dormancy was not observed in the control (E), whose buds began to “germinate” after
ten days.
Plant development was not evident with filter paper plus DDW. The lack of conversion may be
explained mainly by nutrient deficiency and not by the need for hormones. Hormones e.g.,
auxin or cytokinin, reported by Ganapathi et al. (1992), were not added to the SP medium.
Even so, in A and C treatments plant conversion occurred.
Sucrose deficiency was implicated for the lack of ‘germination’ in present
study, green
nodes turned pale yellow or brown after 30 days at 15 °C in the B-D treatments. The lack of
plant conversion (Table 1) is a strong indication that the axillary buds were dead during this
period. Piccioni e Standardi, (1995) considered the nodes to be alive if they could be
maintained green. In our work, the presence of sugar and mineral elements were very
important in the medium for explant survival during storage, e.g.,
A-C treatments and
control (E). By contrast, DDW alone was not sufficient to keep the axillary buds alive for 30
days. In Santalum album tap water as sole source of nutrient in Petri dishes showed a low
percentage of conversion to plants (BAPAT e RAO, 1988). Further research is needed to
assess how sugars and/or mineral nutrition improve the quality of the conversion.
The C treatment was included to compare plant conversion with and without Ca-alginate and
to show that it was not toxic nor depressive (Table 1) The high levels of plant conversion in
C, are in accordance with our routine results in SP medium (BARRUETO CID e CARVALHO,
2008).
The E treatment was a good indicator for vigor and viability of the nodes. When they were
placed in the SP medium at standard conditions, the percent plant conversion of nodes
(axillary buds) after one week was similar to the A treatment. This again showed that the Caalginate did not reduce axillary buds growth in the three genotypes tested.
In conclusion, the use of a Ca-alginate matrix to encapsulate three different cassava genotype
nodes (A treatment) did not depress nor produce toxic effects during the conversion to plants.
All recovered plantlets were without phenotypic variation. On the other hand, low
temperature (15 ◦C) was fundamental to repress the explant grow without to affect axillary
bud survival. Our method enables a simple and practical protocol for the production of
encapsulated nodes from cassava (axillary buds), however, more research are desirable to
evaluate in vitro long-term germplasm conservation at 15 ◦C.
Literature
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encapsulated shoot buds. Plant Cell Reports, Berlin, v. 6, p. 393-395, 1987.
11
BAPAT, V. A; RAO, P. S. Sandalwood plantlets from ‘Synthetic seeds’. Plant Cell Reports,
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