Directory UMM :Data Elmu:jurnal:S:Scientia Horticulturae:Vol83.Issue2.Febr2000:
Scientia Horticulturae 83 (2000) 139±147
Apomictic embryo development and survival in
Uapaca kirkiana under in vitro and
in vivo seed germination
M.F.A. Maliro, M.B. Kwapata*
Bunda College of Agriculture, University of Malawi, PO Box 219, Lilongwe, Malawi
Accepted 6 May 1999
Abstract
In vitro and in vivo studies on seed germination of Uapaca kirkiana were conducted at Bunda
College of Agriculture. In vitro germination treatments included two fruit sources (market and
direct from trees); seed coat (outer and inner layers) removal and two seed germination media
(Murashige and Skoog (MS) and woody plant medium (WPM)). Incubation of in vitro cultures was
under 16 h light at 45 mmol mÿ2 sÿ1 and they were maintained within the range 25±278C. The in
vivo seed germination experiment was set up in a green house with the two fruit sources as main
factors and outer seed coat layer removal and non-removal as sub-factors. Data collection included
number of germinating seeds, number of contaminated cultures and number of normal seedlings per
seed.
Removal of both outer and inner seed coat layers promoted the number of aseptic seedlings. Seed
germinated on MS had a significantly higher frequency of normal seedlings (90%) and multiple
seedlings/seed (90%) than WPM. Removal of the outer seed coat layer improved in vivo
germination from 55% to 78% and from 35% to 95% for market fruits and direct from trees,
respectively. The seeds exhibited apomixis with a maximum of nine seedlings/seed for in vitro, and
two seedlings/seed for in vivo germination. # 2000 Elsevier Science B.V. All rights reserved.
Keywords: Uapaca kirkiana; Tissue culture; Seed germination; Multiple seedlings
* Corresponding author. Tel.: +265-277-222; fax: +265-277-364.
E-mail address: mmaliro@unima.wn.apc.org (M.F.A. Maliro).
0304-4238/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 2 3 8 ( 9 9 ) 0 0 0 7 1 - 0
140
M.F.A. Maliro, M.B. Kwapata / Scientia Horticulturae 83 (2000) 139±147
1. Introduction
Uapaca kirkiana muell. Arg. (Radcliffe Smith, 1993) belongs to the
Euphorbiaceae and is a tropical fruit tree that is indigenous to the miombo
ecozone of Southern Africa (Maghembe and Seyani, 1991; Mwamba, 1995). The
tree bears fruits that are fleshy cream, brown or brownish-red (rufus) in colour,
oval shape, 3±4 cm in diameter with 3±5 seeds (Mwamba, 1995). The fruits are
nutritionally and economically important because of the high nutritive value of
their pulp. They can be eaten raw, made into jam, or used to produce a refreshing
drink and variety of wines like Mulunguzi in Zomba-Malawi and masuku in
Zambia (Kalenga Saka et al., 1989; Kwesiga and Mwanza, 1995). The fruits
collected from the wild are often sold along roadsides and local markets and
significantly contribute to the rural economy. U. kirkiana is a potential
multipurpose tree species for agroforestry practices. It is used for provision of
fuelwood, timber/poles and fruits and can also be exploited for mushroom
production as it is an obligate symbiont of the fungi that form ectomycorrhizae
(Maghembe and Chirwa, 1994).
Despite its value as a potential crop, U. kirkiana has not been domesticated
mainly due to propagation problems. Seed germination under conventional
methods gave poor results (10±37%) after storing the seed at 48C for a prolonged
period because it is recalcitrant (Ngulube and Kananji, 1989). The presence of the
hard outer seed coat layer delays seed germination due to impermeability and
restriction of radicle emergence, which eventually lead to its cotyledons' (leaflike) death. The seed longevity is lost due to pulp rotting and fermenting of juice
with associated production of alcoholic and acidic juices from the sugars in the
pulp which kills the seeds, if extraction of the seeds is delayed (Barton, 1985).
Higher percentage seed germination (43±100%) was achieved with freshly
collected seed when soaked in cold water for 24 h and sown with the outer and
inner seed coat layers completely removed (Prins and Maghembe, 1994). Since
other conventional vegetative propagation methods such as the use of cuttings
have also not succeeded in producing large quantities of U. kirkiana propagules,
it was necessary to investigate alternatives (Kwapata, 1995) such as tissue culture.
The U. kirkiana fruit has 3±5 seeds and each seed coat has two layers. The first
layer (outer layer) is hard and rough and protects the seed from physical damage
and probably insect damage. The second layer (inner layer) is soft and is located
immediately inside the hard outer layer. It is in contact with the two cotyledons
that look like small leaves. The two cotyledons are green in colour when fresh
and alive and supply food to the growing embryo during seed germination. Loss
of the green colour of the cotyledons often indicates that the seed is dead and so
the seeds are always germinated while fresh since they have a short shelf life.
In vitro conditions achieve high germination percentages and provide aseptic
and juvenile plants for rapid micropropagation. In vitro conditions also provide
M.F.A. Maliro, M.B. Kwapata / Scientia Horticulturae 83 (2000) 139±147
141
protection to the seed from infection by microorganisms and from external harsh
conditions. Artificial milleau promotes rapid development of the seedlings which
can be used for rapid multiplication of propagules (Murashige, 1990; Torres,
1988). In vitro germination of most plant seeds is achieved by use of basal salts
medium only. Murashige and Skoog (1962) (MS) formulation is the most
commonly used in most plant tissue cultures, and the alternative is the woody
plant medium (WPM) (McCown and Lloyd, 1981) which was developed
specifically for woody species. Both media were used in the in vitro germination
of U. kirkiana seeds study.
U. kirkiana species is apomictic and seeds would serve as a source of clonal
explants for micropropagation. No study of in vitro germination of U. kirkiana
has been reported so far. This study was therefore initiated to develop a protocol
for in vitro germination of seeds of U. kirkiana in order to improve the
germination and survival of apomictic embryos.
High contamination rates in the initial experiments raised a suspicion that the
fruits obtained from the local market might be infested with fruit fly larvae
(Drosophila sp.) during the ripening process and that these tended to introduce
microorganisms into the innermost parts of the seeds. Such microorganisms in the
internal parts are difficult to eliminate with surface sterilization (Murashige,
1990; Torres, 1988). Infestation might occur because the fruits are not kept under
controlled conditions when in transit from the bush after collection and as they
wait to ripen before being taken to the market. Therefore the source of fruits for
the seed germination experiments was also studied.
The specific objectives of the seed germination experiments were:
1. to determine the U. kirkiana fruit source that gives less contamination in vitro;
2. to determine which of the two basal salts media, Murashige and Skoog (MS)
and woody plant medium (WPM), is better for in vitro U. kirkiana seed
germination; and
3. to evaluate embryo survival rate from U. kirkiana apomictic seed under in
vitro and in vivo conditions.
2. Materials and methods
Experiment 1. In this experiment, the effect of seed coat on the in vitro
germination of U. kirkiana seeds was evaluated. The treatments were (1) both
outer and inner seed coat layers removed and (2) the outer seed coat layer
removed with the inner seed coat layer retained. Ripe fruits were obtained from a
local market (Mitundu) and seeds were extracted. The pulp was washed from the
seeds under tap water and they were then surface sterilized by 2% NaOCl
(commercial sodium hypochlorite) for 20 min. The NaOCl was decanted in a
sterile environment and the seeds were rinsed three times with sterilized distilled
142
M.F.A. Maliro, M.B. Kwapata / Scientia Horticulturae 83 (2000) 139±147
water. MS medium supplemented with 30 g/l sucrose and gelled with 2.5 g/l
phytagel was prepared. The media were dispensed into bellico tubes
(25 mm 150 mm) at 20 ml/tube and sterilized for 10 min under 1218C and
pressure of 100 kps. The media were then transferred to a sterile environment in
an air laminar flow hood where it was allowed to cool and solidify, before
inoculation with seeds. Each treatment had 40 seeds inoculated onto the culture
media at one seed per tube and each tube was covered with kaput and wrapped
with parafilm paper. Incubation room conditions were maintained at 25±278C and
16 h of light daily at 45 mmol mÿ2 sÿ1 provided by white fluorescent tubes. The
following data: number of aseptic cultures, germinating seeds, normal seedlings
(those with health roots and shoots), number of seedlings per seed were collected
weekly for eight weeks.
Experiment 2. A factorial experimental design was used with two U. kirkiana
fruit sources (local market and direct from the trees) as main-factors and two
basal salts media (MS and WPM) as sub-factors. Each treatment combination had
20 cultures. Two basal salts media, MS and WPM were prepared and
supplemented with 16 mg/l White vitamins, 30 g/l sucrose and 10 mg/l of
gentamicin sulphate (an antibiotic) to inhibit development of internal microorganism that might be in the seeds. The media were then adjusted to pH
5.7 0.1 and gelled with 2.5 g/l phytagel. The media were dispensed in bellco
tubes, sterilized and allowed to cool as in Experiment 1.
The outer and inner seed coat layers were removed and the naked seeds were
inoculated onto the medium at one seed/tube. After inoculation each tube was
covered with a kaput and wrapped with parafilm paper. The cultures were
incubated under same conditions as in Experiment 1. The following data: number
of aseptic cultures, germinating seeds, normal seedlings and number of seedlings
per seed were collected weekly for eight weeks.
Experiment 3. A factorial experimental design was used with fruit sources
(local market and direct from trees) as main factors and outer seed coat layer
removal and non-removal as sub-factors. Each treatment combination had 40
seeds. The outer seed coat layer was removed using a surgeon's blade number 10,
scalpels and forceps. The seeds were soaked in distilled water in beakers to
prevent them from dessication before they were sown in two metal trays
(30 cm 40 cm 6 cm) filled with sterilized river sand. Prior to sowing the sand
was saturated with distilled water and eight small-furrows of 1 cm deep were
made in the trays. The seeds without the outer seed coat layer were sown in the
first four furrows and those with intact outer and inner seed coat layers were sown
in the last four furrows. The sowing rate was 10 seeds per furrow. The seeds were
then covered with a thin layer of sand and placed in a humid chamber in the green
house. Moisture was maintained by watering the trays every two days. Data were
collected weekly and included the following: number of seeds emerging, number
of seedlings per seed, were recorded for eight weeks after sowing.
M.F.A. Maliro, M.B. Kwapata / Scientia Horticulturae 83 (2000) 139±147
143
2.1. Calculations and data analysis
The reported parameters were calculated as follows: aseptic cultures
(%) (number of clean seed cultures number of total seed cultures inoculated
(sown) per treatment) 100; seed germination (%) (number of germinating
seeds number of total seeds inoculated (sown) per treatment) 100; normal
seedlings (%) (number of cultures with normal seedlings (with health roots and
shoots) number of total seed cultures inoculated (sown) per treatment) 100;
seedlings with roots only (%) (number of seeds which developed roots only
with no shoots number of totalPseed cultures inoculated per treatment) 100;
root initiation period (days) germination period (days) for seed culture
(ranging from 1 to N) total number of germinated seeds per treatment; and the
calculation procedures for shoot initiation period (days) was the same as for root
initiation period; seeds with multiple seedlings (%) ± (number of germinated
seeds with more than one seedling number of total seed cultures inoculated per
treatment) 100.
Confidence intervals at 95% for successes were calculated using binomial
distribution tables with N 40 or 20 (number of cultures per treatment)
(Snedecor, 1946). Analysis of variance (ANOVA) was performed for number of
seedlings per seed and least significant difference (LSD) for separating the means
was done with Duncan's multiple range test using MSTAT.
3. Results
Experiment 1. The germination percentages were almost within the same range
at 95% confidence limits for seeds with the inner seed coat layer retained and
those with both outer and inner seed coat layers removed. The seeds with both
seed coat layers removed gave a higher percentage of normal (with root and shoot
developed) seedlings (75%) than those with the inner seed coat layer retained
(18%) and fell within different ranges at 95% confidence interval. More aseptic
cultures were obtained from seeds with both seed coat layers removed (78%) than
those with inner seed coat layer retained (43%). There were no significant differences
in time taken from seed inoculation to root or shoot initiation (Table 1).
Experiment 2. There was no significant difference in the number of aseptic
cultures at 95% confidence interval between the fruits from the market and the
tree sources. The MS medium gave significantly (p 0.05) higher percentages of
normal seedlings and seeds with multiple seedlings than the WPM medium
(Table 2). The number of seedlings/seed averaged about 3 and 1 with MS
medium and WPM, respectively, and older fruits from market tended to have
more seedlings per seed than the fresh fruits direct from trees (Table 2). It is
worth noting that the mean values of seedlings per seed were from a data set with
values ranging from 0 to 9 and a standard deviation of 1.8.
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M.F.A. Maliro, M.B. Kwapata / Scientia Horticulturae 83 (2000) 139±147
Table 1
Effect of removal of outer and inner seed coat layers on aseptic seed germination of U. kirkiana
after eight weeks of incubation
Aseptic cultures (%)
Seed germination (%)
Normal seedlings (%)
With roots only (%)
Root initiation period (days)b
Shoot initiation period (days)b
a
b
Inner seed coat
layer retained
Both seed coat
layers removed
43 13a
88 12a
18 9a
20 10a
11
23
78 14a
91 10a
75 13a
16 9a
10
22
Confidence limits at 95%.
Means were not significantly different at p 0.05.
Experiment 3. Removal of the outer seed coat layer resulted in a higher
germination % than without removal for both market and tree fruits. The number
of normal seedlings was not different for market fruits with or without removal of
outer seed coat layer. But tree fruits which retained their outer seed coat layer
produced exceptionally few normal seedlings (28%) when compared with those
that had their outer seed coat layer removed. The number of seeds with multiple
seedlings were similar for the two seed coat treatments regardless of the fruit
source (Table 3). The number of seedlings per seed did not significantly differ
between the two seed coat layer treatments and the fruit sources (Table 3). The
mean values for market and tree fruits were 0.6 and 0.5, respectively, and for
outer seed coat layer removal and non-removal were 0.6 and 0.5, respectively.
These means were from a data set with values ranging from 0 to 2 seedlings per
seed, and a standard deviation of 0.6.
Table 2
Effect of fruit source and basal medium on germination of U. kirkiana seeds after eight weeks of
incubation
Parameter
Source of fruits
Local market (medium)
MS
Aseptic cultures (%)
Normal seedlings (%)
Seeds with multiple seedlings (%)
With roots only (%)
Number of seedlingsb
a
b
Direct from trees (medium)
WPM
a
95 20
90 27a
90 27a
5 5a
3.3
MS
a
100 17
45 22a
35 20a
55 23a
1.3
Confidence limits at 95%.
Means were significantly different at p 0.05; LSD(a0.05)ÿ1.1.
WPM
a
100 17
85 23a
55 23a
10 9a
2.2
100 17a
30 18a
25 19a
65 24a
0.9
145
M.F.A. Maliro, M.B. Kwapata / Scientia Horticulturae 83 (2000) 139±147
Table 3
Effect of outer seed coat layer removal on U. kirkiana seed germination under green house
conditions
Fruit source (S)
Seed germination (%)
Normal seedlings (%)
Seeds with multiple-seedlings (%)
Number of seedlings per seedb
a
b
Local market (outer seed
coat layer treatment)
Direct from trees (outer seed
coat layer treatment)
Removed
Not removed
Removed
Not removed
78 14a
50 14a
5 4a
0.7
55 14a
55 14a
5 4a
0.6
95 9a
43 13a
8 6a
0.5
35 12a
28 13a
10 7a
0.4
Confidence limits at 95%.
Means were not statistically different at p 0.05.
4. Discussion
4.1. In vitro germination of seeds with and without inner seed coat layer
Seeds with inner seed coat layers gave a lower percentage of aseptic cultures
(43%) than those without inner seed coat layers (78%) (Table 1). This implies
that the inner seed coat layers of U. kirkiana seeds harbour some microorganisms
and contamination of seed cultures may be reduced by removal of both the outer
and inner seed coat layers.
There was no significant difference in the seed germination percentage (88%
and 91%), germination period (11 and 10 days) and shoot initiation period (22
and 23 days) for seeds with and without inner seed coat layers (Table 1). These
results suggest that the presence of the inner seed coat layer did not adversely
affect the germination process of the U. kirkiana. However, the development of
seedlings was influenced by the presence of inner seed coat layers since there
were 18% normal seedlings from seeds with inner seed coat layers and 75%
normal seedlings from seeds without the inner seed coat layers. This may
possibly be due to the inner seed coat layers which might have acted as a barrier
between the medium nutrients and the seed embryos leading to poor nutrition of
the developing embryos. On the basis of these results it is recommended that both
outer and inner seed coat layers should be removed to enhance in vitro
germination and normal seedling development of U. kirkiana.
4.2. In vitro germination of seed from different fruit sources and media
The numbers of aseptic cultures were similar in seed germination cultures from
fruits collected either at a local market or direct from the trees, irrespective of the
146
M.F.A. Maliro, M.B. Kwapata / Scientia Horticulturae 83 (2000) 139±147
type of media used (Table 2). However, the MS medium was superior in
producing normal and multiple seedlings than WPM. The differences may be
attributed to the fact that MS medium has a higher concentration of basal salts
than WPM (Huang and Murashige, 1976; Murashige, 1990) and a high nutrient
supply in the medium would promote normal growth of plant tissues. Therefore
MS medium is recommended for use in germination of U. kirkiana seeds under in
vitro conditions.
4.3. Seed germination in greenhouse
In the third experiment, seed germination of U. kirkiana seeds was evaluated
under greenhouse conditions to validate the apomictic behaviour of U. kirkiana
seed exhibited in vitro germination studies where a mean of 3 and a maximum of
9 seedlings per seed were observed.
Removal of the outer seed coat layer improved germination from 55% to
78% and 35% to 95% for fruits from the market and direct from trees,
respectively, under the green-house conditions. Multiple seedlings were also
observed in seeds germinated in a greenhouse at the frequency range 5±10% of
the total number of seeds planted and the number of seedlings/seed were not
more than two.
As in the in vitro study the in vivo results also showed multiple seedlings
developing from U. kirkiana seed during germination. Therefore the polyembryony (apomixis) tendency may not be attributed to the in vitro conditions alone,
but to inherent characteristics of the U. kirkiana seed itself. However, the
development and survival of apomictic embryos vary under in vitro and in vivo
conditions. In vitro, the number of seedlings per seed ranged from 1 to 9 with a
mean of 3 and a standard deviation of 1.8, while in vivo, seedlings per seed
ranged from 0 to 25 with a mean of 1 and standard deviation of 0.6. These results,
strongly suggest that the development and survival of apomictic embryos in U.
kirkiana is enhanced by in vitro conditions. The fact that in apomixis, all the
seedlings, except one, are asexual and true to type of the parental trees (Esau,
1977; Hartmann and Kester, 1975) the apomictic property of the seed of U.
kirkiana can be exploited as a source of clonal explants for the rapid
multiplication of propagules. However, the problem is the accurate identification
of the apomictic seedlings, and further study is recommended to develop criteria
for selection of asexual from sexual seedlings.
Acknowledgements
We thank the International Centre for Research in Agroforestry (ICRAF) for its
financial support.
M.F.A. Maliro, M.B. Kwapata / Scientia Horticulturae 83 (2000) 139±147
147
References
Barton, L.V., 1985. Seed Preservation and Longevity. International Books and Periodicals Supply
Service, New Delhi, 216 p.
Esau, K., 1977. Anatomy of Seed Plants, 2nd ed. Wiley, New York, 550 pp.
Hartmann, H.T., Kester, D.E., 1975. Plant Propagation ± Principles and Practices, 3rd ed. PrenticeHall, Englewood Cliffs, NJ, 662 pp.
Huang, L., Murashige, T., 1976. Plant tissue culture media: major constituents, their preparation
and some applications. Tissue Culture Manual, vol. 3, no. 1. Rockville, USA, pp. 539±548.
Kalenga Saka, J.D., Msonthi, J.D., Sambo, E.Y., 1989. Ascorbic and mineral contents of edible wild
fruits growing in Malawi. In: Kwapata, M.B., Kanyama Phiri, G.Y., Kasomekera, Z. (Eds.),
Proceedings of the XIIth Annual Conference on Science and Technology for Development,
AASToM held in Blantyre, 17±19 December 1989, pp. 98±103.
Kwapata, M., 1995. The potential use of tissue culture in the propagation of indigenous fruit trees.
In: Maghembe, J.A., Ntupanyama, Y., Chirwa, P.W. (Eds.), Improvement of Indigenous Fruit
Trees of the Miombo Woodlands of southern Africa. Primex Printers, ICRAF, Nairobi, pp. 73±
77.
Kwesiga, F., Mwanza, S., 1995. Underexploited wild genetic resources: the case of indigenous fruit
trees in eastern Zambia. In: Maghembe, J.A., Ntupanyama, Y., Chirwa, P.W. (Eds.),
Improvement of Indigenous Fruit Trees of the Miombo Woodlands of southern Africa. Primex
Printers, ICRAF, Nairobi, pp. 100±112.
Maghembe, J.A., Chirwa, P.W., 1994. Malawi SADC/ICRAF Agroforestry Project Annual Research
Report for Year Ending, October 1994, Zomba, Malawi.
Maghembe, J.A., Seyani, J.H., 1991. Multipurpose trees used by smallholder farmers in Malawi:
results of an ethnobotanical survey. Agroforestry Research Networks for Africa (AFRENA)
Report No. 42, 30 pp.
McCown, B.H., Lloyd, G., 1981. Woody plant medium (WPM) ± a mineral nutrient formulation for
microculture for woody plant species. HortScience 16, 453.
Murashige, T., 1990. Plant propagation by tissue culture: a practice with unrealized potential. In:
Ammirato, P.V., Evans, D.A., Sharp, W.R., Bajaj, Y.P.S. (Eds.), Handbook of Plant Cell Culture,
vol. 5, McGraw-Hill, New York, pp. 3±9.
Murashige, T., Skoog, F., 1962. Revised medium for rapid growth and bioassays with tobacco tissue
cultures. Physiol. Plant 15, 473±497.
Mwamba, C.K., 1995. Variations in fruit of Uapaca kirkiana and effects of in situ silvicultural
treatments on fruit parameters. In: Maghembe, J.A., Ntupanyama, Y., Chirwa, P.W. (Eds.),
Improvement of Indigenous Fruit Trees of the Miombo Woodlands of southern Africa. Primex
Printers, ICRAF, Nairobi, pp. 27±38.
Ngulube, M.R., Kananji, B., 1989. Seed problems of edible indigenous fruits in Malawi. In:
Kwapata, M.B., Kanyama Phiri, G.Y., Kasomekera, Z. (Eds.), Proceedings of the XIIth Annual
Conference on Science and Technology for Development, AASToM held in Blantyre, 17±19
December 1989, pp. 104±109.
Prins, H., Maghembe, J.A., 1994. Germination studies on seed of fruit trees indigenous to Malawi.
Forest Ecology and Management 64, 111±125.
Radcliffe Smith, A., 1993. Notes on African Euphorbiaceae XXIX: Uapaca. Kew Bulletin, vol. 48,
no. 3, pp. 611±617.
Snedecor, G.W., 1946. Statistical Methods, 4th ed. Iowa State College Press, Ames.
Torres, K.C., 1988. Tissue Culture Techniques for Horticultural Crops. Van Nostrand Reinhold,
New York, pp. 26±48.
Apomictic embryo development and survival in
Uapaca kirkiana under in vitro and
in vivo seed germination
M.F.A. Maliro, M.B. Kwapata*
Bunda College of Agriculture, University of Malawi, PO Box 219, Lilongwe, Malawi
Accepted 6 May 1999
Abstract
In vitro and in vivo studies on seed germination of Uapaca kirkiana were conducted at Bunda
College of Agriculture. In vitro germination treatments included two fruit sources (market and
direct from trees); seed coat (outer and inner layers) removal and two seed germination media
(Murashige and Skoog (MS) and woody plant medium (WPM)). Incubation of in vitro cultures was
under 16 h light at 45 mmol mÿ2 sÿ1 and they were maintained within the range 25±278C. The in
vivo seed germination experiment was set up in a green house with the two fruit sources as main
factors and outer seed coat layer removal and non-removal as sub-factors. Data collection included
number of germinating seeds, number of contaminated cultures and number of normal seedlings per
seed.
Removal of both outer and inner seed coat layers promoted the number of aseptic seedlings. Seed
germinated on MS had a significantly higher frequency of normal seedlings (90%) and multiple
seedlings/seed (90%) than WPM. Removal of the outer seed coat layer improved in vivo
germination from 55% to 78% and from 35% to 95% for market fruits and direct from trees,
respectively. The seeds exhibited apomixis with a maximum of nine seedlings/seed for in vitro, and
two seedlings/seed for in vivo germination. # 2000 Elsevier Science B.V. All rights reserved.
Keywords: Uapaca kirkiana; Tissue culture; Seed germination; Multiple seedlings
* Corresponding author. Tel.: +265-277-222; fax: +265-277-364.
E-mail address: mmaliro@unima.wn.apc.org (M.F.A. Maliro).
0304-4238/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 2 3 8 ( 9 9 ) 0 0 0 7 1 - 0
140
M.F.A. Maliro, M.B. Kwapata / Scientia Horticulturae 83 (2000) 139±147
1. Introduction
Uapaca kirkiana muell. Arg. (Radcliffe Smith, 1993) belongs to the
Euphorbiaceae and is a tropical fruit tree that is indigenous to the miombo
ecozone of Southern Africa (Maghembe and Seyani, 1991; Mwamba, 1995). The
tree bears fruits that are fleshy cream, brown or brownish-red (rufus) in colour,
oval shape, 3±4 cm in diameter with 3±5 seeds (Mwamba, 1995). The fruits are
nutritionally and economically important because of the high nutritive value of
their pulp. They can be eaten raw, made into jam, or used to produce a refreshing
drink and variety of wines like Mulunguzi in Zomba-Malawi and masuku in
Zambia (Kalenga Saka et al., 1989; Kwesiga and Mwanza, 1995). The fruits
collected from the wild are often sold along roadsides and local markets and
significantly contribute to the rural economy. U. kirkiana is a potential
multipurpose tree species for agroforestry practices. It is used for provision of
fuelwood, timber/poles and fruits and can also be exploited for mushroom
production as it is an obligate symbiont of the fungi that form ectomycorrhizae
(Maghembe and Chirwa, 1994).
Despite its value as a potential crop, U. kirkiana has not been domesticated
mainly due to propagation problems. Seed germination under conventional
methods gave poor results (10±37%) after storing the seed at 48C for a prolonged
period because it is recalcitrant (Ngulube and Kananji, 1989). The presence of the
hard outer seed coat layer delays seed germination due to impermeability and
restriction of radicle emergence, which eventually lead to its cotyledons' (leaflike) death. The seed longevity is lost due to pulp rotting and fermenting of juice
with associated production of alcoholic and acidic juices from the sugars in the
pulp which kills the seeds, if extraction of the seeds is delayed (Barton, 1985).
Higher percentage seed germination (43±100%) was achieved with freshly
collected seed when soaked in cold water for 24 h and sown with the outer and
inner seed coat layers completely removed (Prins and Maghembe, 1994). Since
other conventional vegetative propagation methods such as the use of cuttings
have also not succeeded in producing large quantities of U. kirkiana propagules,
it was necessary to investigate alternatives (Kwapata, 1995) such as tissue culture.
The U. kirkiana fruit has 3±5 seeds and each seed coat has two layers. The first
layer (outer layer) is hard and rough and protects the seed from physical damage
and probably insect damage. The second layer (inner layer) is soft and is located
immediately inside the hard outer layer. It is in contact with the two cotyledons
that look like small leaves. The two cotyledons are green in colour when fresh
and alive and supply food to the growing embryo during seed germination. Loss
of the green colour of the cotyledons often indicates that the seed is dead and so
the seeds are always germinated while fresh since they have a short shelf life.
In vitro conditions achieve high germination percentages and provide aseptic
and juvenile plants for rapid micropropagation. In vitro conditions also provide
M.F.A. Maliro, M.B. Kwapata / Scientia Horticulturae 83 (2000) 139±147
141
protection to the seed from infection by microorganisms and from external harsh
conditions. Artificial milleau promotes rapid development of the seedlings which
can be used for rapid multiplication of propagules (Murashige, 1990; Torres,
1988). In vitro germination of most plant seeds is achieved by use of basal salts
medium only. Murashige and Skoog (1962) (MS) formulation is the most
commonly used in most plant tissue cultures, and the alternative is the woody
plant medium (WPM) (McCown and Lloyd, 1981) which was developed
specifically for woody species. Both media were used in the in vitro germination
of U. kirkiana seeds study.
U. kirkiana species is apomictic and seeds would serve as a source of clonal
explants for micropropagation. No study of in vitro germination of U. kirkiana
has been reported so far. This study was therefore initiated to develop a protocol
for in vitro germination of seeds of U. kirkiana in order to improve the
germination and survival of apomictic embryos.
High contamination rates in the initial experiments raised a suspicion that the
fruits obtained from the local market might be infested with fruit fly larvae
(Drosophila sp.) during the ripening process and that these tended to introduce
microorganisms into the innermost parts of the seeds. Such microorganisms in the
internal parts are difficult to eliminate with surface sterilization (Murashige,
1990; Torres, 1988). Infestation might occur because the fruits are not kept under
controlled conditions when in transit from the bush after collection and as they
wait to ripen before being taken to the market. Therefore the source of fruits for
the seed germination experiments was also studied.
The specific objectives of the seed germination experiments were:
1. to determine the U. kirkiana fruit source that gives less contamination in vitro;
2. to determine which of the two basal salts media, Murashige and Skoog (MS)
and woody plant medium (WPM), is better for in vitro U. kirkiana seed
germination; and
3. to evaluate embryo survival rate from U. kirkiana apomictic seed under in
vitro and in vivo conditions.
2. Materials and methods
Experiment 1. In this experiment, the effect of seed coat on the in vitro
germination of U. kirkiana seeds was evaluated. The treatments were (1) both
outer and inner seed coat layers removed and (2) the outer seed coat layer
removed with the inner seed coat layer retained. Ripe fruits were obtained from a
local market (Mitundu) and seeds were extracted. The pulp was washed from the
seeds under tap water and they were then surface sterilized by 2% NaOCl
(commercial sodium hypochlorite) for 20 min. The NaOCl was decanted in a
sterile environment and the seeds were rinsed three times with sterilized distilled
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water. MS medium supplemented with 30 g/l sucrose and gelled with 2.5 g/l
phytagel was prepared. The media were dispensed into bellico tubes
(25 mm 150 mm) at 20 ml/tube and sterilized for 10 min under 1218C and
pressure of 100 kps. The media were then transferred to a sterile environment in
an air laminar flow hood where it was allowed to cool and solidify, before
inoculation with seeds. Each treatment had 40 seeds inoculated onto the culture
media at one seed per tube and each tube was covered with kaput and wrapped
with parafilm paper. Incubation room conditions were maintained at 25±278C and
16 h of light daily at 45 mmol mÿ2 sÿ1 provided by white fluorescent tubes. The
following data: number of aseptic cultures, germinating seeds, normal seedlings
(those with health roots and shoots), number of seedlings per seed were collected
weekly for eight weeks.
Experiment 2. A factorial experimental design was used with two U. kirkiana
fruit sources (local market and direct from the trees) as main-factors and two
basal salts media (MS and WPM) as sub-factors. Each treatment combination had
20 cultures. Two basal salts media, MS and WPM were prepared and
supplemented with 16 mg/l White vitamins, 30 g/l sucrose and 10 mg/l of
gentamicin sulphate (an antibiotic) to inhibit development of internal microorganism that might be in the seeds. The media were then adjusted to pH
5.7 0.1 and gelled with 2.5 g/l phytagel. The media were dispensed in bellco
tubes, sterilized and allowed to cool as in Experiment 1.
The outer and inner seed coat layers were removed and the naked seeds were
inoculated onto the medium at one seed/tube. After inoculation each tube was
covered with a kaput and wrapped with parafilm paper. The cultures were
incubated under same conditions as in Experiment 1. The following data: number
of aseptic cultures, germinating seeds, normal seedlings and number of seedlings
per seed were collected weekly for eight weeks.
Experiment 3. A factorial experimental design was used with fruit sources
(local market and direct from trees) as main factors and outer seed coat layer
removal and non-removal as sub-factors. Each treatment combination had 40
seeds. The outer seed coat layer was removed using a surgeon's blade number 10,
scalpels and forceps. The seeds were soaked in distilled water in beakers to
prevent them from dessication before they were sown in two metal trays
(30 cm 40 cm 6 cm) filled with sterilized river sand. Prior to sowing the sand
was saturated with distilled water and eight small-furrows of 1 cm deep were
made in the trays. The seeds without the outer seed coat layer were sown in the
first four furrows and those with intact outer and inner seed coat layers were sown
in the last four furrows. The sowing rate was 10 seeds per furrow. The seeds were
then covered with a thin layer of sand and placed in a humid chamber in the green
house. Moisture was maintained by watering the trays every two days. Data were
collected weekly and included the following: number of seeds emerging, number
of seedlings per seed, were recorded for eight weeks after sowing.
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143
2.1. Calculations and data analysis
The reported parameters were calculated as follows: aseptic cultures
(%) (number of clean seed cultures number of total seed cultures inoculated
(sown) per treatment) 100; seed germination (%) (number of germinating
seeds number of total seeds inoculated (sown) per treatment) 100; normal
seedlings (%) (number of cultures with normal seedlings (with health roots and
shoots) number of total seed cultures inoculated (sown) per treatment) 100;
seedlings with roots only (%) (number of seeds which developed roots only
with no shoots number of totalPseed cultures inoculated per treatment) 100;
root initiation period (days) germination period (days) for seed culture
(ranging from 1 to N) total number of germinated seeds per treatment; and the
calculation procedures for shoot initiation period (days) was the same as for root
initiation period; seeds with multiple seedlings (%) ± (number of germinated
seeds with more than one seedling number of total seed cultures inoculated per
treatment) 100.
Confidence intervals at 95% for successes were calculated using binomial
distribution tables with N 40 or 20 (number of cultures per treatment)
(Snedecor, 1946). Analysis of variance (ANOVA) was performed for number of
seedlings per seed and least significant difference (LSD) for separating the means
was done with Duncan's multiple range test using MSTAT.
3. Results
Experiment 1. The germination percentages were almost within the same range
at 95% confidence limits for seeds with the inner seed coat layer retained and
those with both outer and inner seed coat layers removed. The seeds with both
seed coat layers removed gave a higher percentage of normal (with root and shoot
developed) seedlings (75%) than those with the inner seed coat layer retained
(18%) and fell within different ranges at 95% confidence interval. More aseptic
cultures were obtained from seeds with both seed coat layers removed (78%) than
those with inner seed coat layer retained (43%). There were no significant differences
in time taken from seed inoculation to root or shoot initiation (Table 1).
Experiment 2. There was no significant difference in the number of aseptic
cultures at 95% confidence interval between the fruits from the market and the
tree sources. The MS medium gave significantly (p 0.05) higher percentages of
normal seedlings and seeds with multiple seedlings than the WPM medium
(Table 2). The number of seedlings/seed averaged about 3 and 1 with MS
medium and WPM, respectively, and older fruits from market tended to have
more seedlings per seed than the fresh fruits direct from trees (Table 2). It is
worth noting that the mean values of seedlings per seed were from a data set with
values ranging from 0 to 9 and a standard deviation of 1.8.
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Table 1
Effect of removal of outer and inner seed coat layers on aseptic seed germination of U. kirkiana
after eight weeks of incubation
Aseptic cultures (%)
Seed germination (%)
Normal seedlings (%)
With roots only (%)
Root initiation period (days)b
Shoot initiation period (days)b
a
b
Inner seed coat
layer retained
Both seed coat
layers removed
43 13a
88 12a
18 9a
20 10a
11
23
78 14a
91 10a
75 13a
16 9a
10
22
Confidence limits at 95%.
Means were not significantly different at p 0.05.
Experiment 3. Removal of the outer seed coat layer resulted in a higher
germination % than without removal for both market and tree fruits. The number
of normal seedlings was not different for market fruits with or without removal of
outer seed coat layer. But tree fruits which retained their outer seed coat layer
produced exceptionally few normal seedlings (28%) when compared with those
that had their outer seed coat layer removed. The number of seeds with multiple
seedlings were similar for the two seed coat treatments regardless of the fruit
source (Table 3). The number of seedlings per seed did not significantly differ
between the two seed coat layer treatments and the fruit sources (Table 3). The
mean values for market and tree fruits were 0.6 and 0.5, respectively, and for
outer seed coat layer removal and non-removal were 0.6 and 0.5, respectively.
These means were from a data set with values ranging from 0 to 2 seedlings per
seed, and a standard deviation of 0.6.
Table 2
Effect of fruit source and basal medium on germination of U. kirkiana seeds after eight weeks of
incubation
Parameter
Source of fruits
Local market (medium)
MS
Aseptic cultures (%)
Normal seedlings (%)
Seeds with multiple seedlings (%)
With roots only (%)
Number of seedlingsb
a
b
Direct from trees (medium)
WPM
a
95 20
90 27a
90 27a
5 5a
3.3
MS
a
100 17
45 22a
35 20a
55 23a
1.3
Confidence limits at 95%.
Means were significantly different at p 0.05; LSD(a0.05)ÿ1.1.
WPM
a
100 17
85 23a
55 23a
10 9a
2.2
100 17a
30 18a
25 19a
65 24a
0.9
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M.F.A. Maliro, M.B. Kwapata / Scientia Horticulturae 83 (2000) 139±147
Table 3
Effect of outer seed coat layer removal on U. kirkiana seed germination under green house
conditions
Fruit source (S)
Seed germination (%)
Normal seedlings (%)
Seeds with multiple-seedlings (%)
Number of seedlings per seedb
a
b
Local market (outer seed
coat layer treatment)
Direct from trees (outer seed
coat layer treatment)
Removed
Not removed
Removed
Not removed
78 14a
50 14a
5 4a
0.7
55 14a
55 14a
5 4a
0.6
95 9a
43 13a
8 6a
0.5
35 12a
28 13a
10 7a
0.4
Confidence limits at 95%.
Means were not statistically different at p 0.05.
4. Discussion
4.1. In vitro germination of seeds with and without inner seed coat layer
Seeds with inner seed coat layers gave a lower percentage of aseptic cultures
(43%) than those without inner seed coat layers (78%) (Table 1). This implies
that the inner seed coat layers of U. kirkiana seeds harbour some microorganisms
and contamination of seed cultures may be reduced by removal of both the outer
and inner seed coat layers.
There was no significant difference in the seed germination percentage (88%
and 91%), germination period (11 and 10 days) and shoot initiation period (22
and 23 days) for seeds with and without inner seed coat layers (Table 1). These
results suggest that the presence of the inner seed coat layer did not adversely
affect the germination process of the U. kirkiana. However, the development of
seedlings was influenced by the presence of inner seed coat layers since there
were 18% normal seedlings from seeds with inner seed coat layers and 75%
normal seedlings from seeds without the inner seed coat layers. This may
possibly be due to the inner seed coat layers which might have acted as a barrier
between the medium nutrients and the seed embryos leading to poor nutrition of
the developing embryos. On the basis of these results it is recommended that both
outer and inner seed coat layers should be removed to enhance in vitro
germination and normal seedling development of U. kirkiana.
4.2. In vitro germination of seed from different fruit sources and media
The numbers of aseptic cultures were similar in seed germination cultures from
fruits collected either at a local market or direct from the trees, irrespective of the
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M.F.A. Maliro, M.B. Kwapata / Scientia Horticulturae 83 (2000) 139±147
type of media used (Table 2). However, the MS medium was superior in
producing normal and multiple seedlings than WPM. The differences may be
attributed to the fact that MS medium has a higher concentration of basal salts
than WPM (Huang and Murashige, 1976; Murashige, 1990) and a high nutrient
supply in the medium would promote normal growth of plant tissues. Therefore
MS medium is recommended for use in germination of U. kirkiana seeds under in
vitro conditions.
4.3. Seed germination in greenhouse
In the third experiment, seed germination of U. kirkiana seeds was evaluated
under greenhouse conditions to validate the apomictic behaviour of U. kirkiana
seed exhibited in vitro germination studies where a mean of 3 and a maximum of
9 seedlings per seed were observed.
Removal of the outer seed coat layer improved germination from 55% to
78% and 35% to 95% for fruits from the market and direct from trees,
respectively, under the green-house conditions. Multiple seedlings were also
observed in seeds germinated in a greenhouse at the frequency range 5±10% of
the total number of seeds planted and the number of seedlings/seed were not
more than two.
As in the in vitro study the in vivo results also showed multiple seedlings
developing from U. kirkiana seed during germination. Therefore the polyembryony (apomixis) tendency may not be attributed to the in vitro conditions alone,
but to inherent characteristics of the U. kirkiana seed itself. However, the
development and survival of apomictic embryos vary under in vitro and in vivo
conditions. In vitro, the number of seedlings per seed ranged from 1 to 9 with a
mean of 3 and a standard deviation of 1.8, while in vivo, seedlings per seed
ranged from 0 to 25 with a mean of 1 and standard deviation of 0.6. These results,
strongly suggest that the development and survival of apomictic embryos in U.
kirkiana is enhanced by in vitro conditions. The fact that in apomixis, all the
seedlings, except one, are asexual and true to type of the parental trees (Esau,
1977; Hartmann and Kester, 1975) the apomictic property of the seed of U.
kirkiana can be exploited as a source of clonal explants for the rapid
multiplication of propagules. However, the problem is the accurate identification
of the apomictic seedlings, and further study is recommended to develop criteria
for selection of asexual from sexual seedlings.
Acknowledgements
We thank the International Centre for Research in Agroforestry (ICRAF) for its
financial support.
M.F.A. Maliro, M.B. Kwapata / Scientia Horticulturae 83 (2000) 139±147
147
References
Barton, L.V., 1985. Seed Preservation and Longevity. International Books and Periodicals Supply
Service, New Delhi, 216 p.
Esau, K., 1977. Anatomy of Seed Plants, 2nd ed. Wiley, New York, 550 pp.
Hartmann, H.T., Kester, D.E., 1975. Plant Propagation ± Principles and Practices, 3rd ed. PrenticeHall, Englewood Cliffs, NJ, 662 pp.
Huang, L., Murashige, T., 1976. Plant tissue culture media: major constituents, their preparation
and some applications. Tissue Culture Manual, vol. 3, no. 1. Rockville, USA, pp. 539±548.
Kalenga Saka, J.D., Msonthi, J.D., Sambo, E.Y., 1989. Ascorbic and mineral contents of edible wild
fruits growing in Malawi. In: Kwapata, M.B., Kanyama Phiri, G.Y., Kasomekera, Z. (Eds.),
Proceedings of the XIIth Annual Conference on Science and Technology for Development,
AASToM held in Blantyre, 17±19 December 1989, pp. 98±103.
Kwapata, M., 1995. The potential use of tissue culture in the propagation of indigenous fruit trees.
In: Maghembe, J.A., Ntupanyama, Y., Chirwa, P.W. (Eds.), Improvement of Indigenous Fruit
Trees of the Miombo Woodlands of southern Africa. Primex Printers, ICRAF, Nairobi, pp. 73±
77.
Kwesiga, F., Mwanza, S., 1995. Underexploited wild genetic resources: the case of indigenous fruit
trees in eastern Zambia. In: Maghembe, J.A., Ntupanyama, Y., Chirwa, P.W. (Eds.),
Improvement of Indigenous Fruit Trees of the Miombo Woodlands of southern Africa. Primex
Printers, ICRAF, Nairobi, pp. 100±112.
Maghembe, J.A., Chirwa, P.W., 1994. Malawi SADC/ICRAF Agroforestry Project Annual Research
Report for Year Ending, October 1994, Zomba, Malawi.
Maghembe, J.A., Seyani, J.H., 1991. Multipurpose trees used by smallholder farmers in Malawi:
results of an ethnobotanical survey. Agroforestry Research Networks for Africa (AFRENA)
Report No. 42, 30 pp.
McCown, B.H., Lloyd, G., 1981. Woody plant medium (WPM) ± a mineral nutrient formulation for
microculture for woody plant species. HortScience 16, 453.
Murashige, T., 1990. Plant propagation by tissue culture: a practice with unrealized potential. In:
Ammirato, P.V., Evans, D.A., Sharp, W.R., Bajaj, Y.P.S. (Eds.), Handbook of Plant Cell Culture,
vol. 5, McGraw-Hill, New York, pp. 3±9.
Murashige, T., Skoog, F., 1962. Revised medium for rapid growth and bioassays with tobacco tissue
cultures. Physiol. Plant 15, 473±497.
Mwamba, C.K., 1995. Variations in fruit of Uapaca kirkiana and effects of in situ silvicultural
treatments on fruit parameters. In: Maghembe, J.A., Ntupanyama, Y., Chirwa, P.W. (Eds.),
Improvement of Indigenous Fruit Trees of the Miombo Woodlands of southern Africa. Primex
Printers, ICRAF, Nairobi, pp. 27±38.
Ngulube, M.R., Kananji, B., 1989. Seed problems of edible indigenous fruits in Malawi. In:
Kwapata, M.B., Kanyama Phiri, G.Y., Kasomekera, Z. (Eds.), Proceedings of the XIIth Annual
Conference on Science and Technology for Development, AASToM held in Blantyre, 17±19
December 1989, pp. 104±109.
Prins, H., Maghembe, J.A., 1994. Germination studies on seed of fruit trees indigenous to Malawi.
Forest Ecology and Management 64, 111±125.
Radcliffe Smith, A., 1993. Notes on African Euphorbiaceae XXIX: Uapaca. Kew Bulletin, vol. 48,
no. 3, pp. 611±617.
Snedecor, G.W., 1946. Statistical Methods, 4th ed. Iowa State College Press, Ames.
Torres, K.C., 1988. Tissue Culture Techniques for Horticultural Crops. Van Nostrand Reinhold,
New York, pp. 26±48.