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 ofUapaca kirkianawere 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 45mmol mÿ2sÿ1and 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).
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' (leaf-like) 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 ofU. kirkianapropagules, it was necessary to investigate alternatives (Kwapata, 1995) such as tissue culture. TheU. kirkianafruit 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.
protection to the seed from infection by microorganisms and from external harsh conditions. Artificialmilleaupromotes 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 ofU. kirkianaseeds 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 theU. kirkianafruit 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
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 mm150 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 45mmol mÿ2sÿ1 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.
Experiment2. A factorial experimental design was used with twoU. 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 micro-organism that might be in the seeds. The media were then adjusted to pH 5.70.1 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.
2.1. Calculations and data analysis
The reported parameters were calculated as follows: aseptic cultures (%)(number of clean seed culturesnumber of total seed cultures inoculated (sown) per treatment)100; seed germination (%)(number of germinating seedsnumber 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 shootsnumber of total seed cultures inoculated per treatment)100; root initiation period (days)Pgermination period (days) for seed culture (ranging from 1 toN)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 seedlingnumber of total seed cultures inoculated per treatment)100.
Confidence intervals at 95% for successes were calculated using binomial distribution tables with N40 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
Experiment1. 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 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 of0.6.
Table 1
Effect of removal of outer and inner seed coat layers on aseptic seed germination ofU. kirkiana
after eight weeks of incubation
Inner seed coat layer retained
Both seed coat layers removed
Aseptic cultures (%) 4313a 7814a
Seed germination (%) 8812a 9110a
Normal seedlings (%) 189a 7513a
With roots only (%) 2010a 169a
Root initiation period (days)b 11 10
Shoot initiation period (days)b 23 22
a
Confidence limits at 95%.
b
Means were not significantly different atp0.05.
Table 2
Effect of fruit source and basal medium on germination ofU. kirkianaseeds after eight weeks of incubation
Parameter Source of fruits
Local market (medium) Direct from trees (medium)
MS WPM MS WPM
Aseptic cultures (%) 9520a 10017a 10017a 10017a Normal seedlings (%) 9027a 4522a 8523a 3018a Seeds with multiple seedlings (%) 9027a 3520a 5523a 2519a With roots only (%) 55a 5523a 109a 6524a
Number of seedlingsb 3.3 1.3 2.2 0.9
a
Confidence limits at 95%.
bMeans were significantly different at
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 ofU. kirkianaseeds 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 ofU. 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 Table 3
Effect of outer seed coat layer removal on U. kirkiana seed germination under green house conditions
Fruit source (S)
Local market (outer seed coat layer treatment)
Direct from trees (outer seed coat layer treatment)
Removed Not removed Removed Not removed
Seed germination (%) 7814a 5514a 959a 3512a Normal seedlings (%) 5014a 5514a 4313a 2813a Seeds with multiple-seedlings (%) 54a 54a 86a 107a Number of seedlings per seedb 0.7 0.6 0.5 0.4
a
Confidence limits at 95%.
bMeans were not statistically different at
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 ofU. kirkianaseeds underin 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 ofU. kirkiana seed exhibitedin vitrogermination 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 polyem-bryony (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 inU. 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
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