- Email: [email protected]
In vitro propagation of Kniphofia pauciflora Bak. for conservation purposes
S. Afr. J. Bot. 1996, 62(4): 219-22 1
2I9
leaf anatomical evidence for regarding Chortolirion as a monotype, it is proposed that the genus should still be maintained separate from Haworthia. This stems primarily from its bulbous vegetative morphology and acuminate capsules, both features being absent in Haworrhia. Futhermore, it is the only haworthioid species of which the leaves are deciduous and die back to ground level after !ires or frost. It is, however, clear that the outer periclinal epidermal cell walls of Clwrtolirion are strongly PAS-positive, whereas, in H. blackbumiae and H. graminifolia, the corresponding walls are only slightly so (Figures 1-3), indicating a general lack of complex polysaccharides in the graminoid haworthias. A difference in fixatives could not have been a causal factor; the epidermal layers of H. kingiana and H. koe/maniorum showed PAS-positivity after treatment with the same fixative as the graminoid haworthias (compare Figures 5 & 6 with Figures 2 & 3). Although this featu re could be an additional diagnostic character for separating Chortolirion from grass-leaved species of Haworthia, a more comprehensive and detailed study is required before inferences can be made of its taxonomic value.
In vitro propagation of Kniphofia pauciflora Bak. for conservation purposes
Acknowledgements Mr Ian Oliver, curator of the Karoo National Botanical Garden, Worcester, is thanked for providing material of H. blackbumiae and H. graminifolia.
Keywords: Indigenous, Kniphofia pauclflora, micropropagation, rare, tissue culture.
References BAYER. M.B. 1972. Reinstatement of the genus A.mo/oba and Poe/1nitzia Uitw. (Liliaceae-Aioineae). Nat. Cact. Scux. J. 27: 77-79. COLLINS, B.A. & MACNICHOL. E.F. Uun.) 1978. Long term fixation for histological studies. Mkms,·. Al."ta 81: 155-158. FEDER. N. & O'BRIEN. T.P. 1968. Plant microtechnique: some principles and new methods. Am. J. Bot. 55: 123- 142. HILTON-TAY LOR. C. & SMITH. G.F. 1994. The conservation status of Aloaccae 111 southern Africa. In: Botanical diversity in southern Africa, cd. B.J. Huntley. pp. 287-303. Stre/iczia l. National Botanical Institute, Prctona. MUELLER. W.C. & GREENWOOD. A.D. 1977. The ultrastructure of phenolic cells lixed with caffeine. J. exp. Bot. 29: 757-764. SMITH. G.F. 1990. Nomenclatural notes on the subsection Bowieae in Aloe (Asphodeluceae: Alooideae). S. Afr. J. Bot. 56: 303-308. SM ITH. G.F. 199 1. Studies on the reproductive biology and palynology of Clwrtoliritm Berger (Asphodelaccae: Aloaceae) in southern Africn. Taxon40 : 61-73. SMITH. G.F. 1994. Taxonomic history of Poellnitzia Uitcwaal, a unispccific genus of Alooideae (Asphodelaceae). Haseltonia 2: 74-78. SMITH. G.F. & VAN WYK. A.E. 1992. Systematic leaf anatomy of selected genera of southern African Alooideae (Asphodelaceae). S. A.(1: 1. Bot. 58: 349- 357.543. SMITH. G.F. & VANWYK. A.E. 1993. The generic status of Chortolirion (Aioaccnc). Kt•w Bull. 48: lOS-I 13. SM ITH. G.F. & VAN WYK. B-E. I991. Generic relationships in the Alooideac (Asphodelacene). Taxon 40: 557-581.
Kniphofia paucijlora Bak. is a pretty plant belonging to the subfamily Asphodeloideae (Dahlgren et al. 1985). The plants have short stolons, forming groups of short stems. The 20-35-cm leaves are soft and yellow green. The leaves are 2- 8 mm wide and are triangular in cross section (Codd 1968). The flower spike is 30-50 em long and is raised above the leaves. The inflorescence is yellow and few-flowered compared to other Kniphojia sp. (Figure I A). These plants flower from September to November. Kniphojia paucijlora is known only from restricted areas near Durban in Natal, where it used to grow in marshy grasslands. This species is threatened due to urbanization and is no longer found at the type locality. Specimens were found in the Marian Hill area in 1951 and I 954 but the habitat has since been drained and the plants have disappeared from this region (Codd 1968). Conservation of this plant is needed and tissue culture was undertaken to increase the limited numbers held in the National Botanical Garden in Pietermaritzburg. Only one reference to the in vitro culture of Kniphofia species could be found (Nayak & Sen I 990). A single plant was obtained from the Botanical Garden in Pietermaritzburg to use as explant source. First attempts at culturing this plant were undertaken using leaf and peduncle explants. The material was sterilized for 5 and 10 min using 3.5% sodium hypochlorite and then placed on Murashige and Skoog (1962) medium (MS) solidified with 0.8% Unilab agar. The pH was adjusted to 5.8 with I M NaOH prior to being autoclaved. 1-Naphthaieneacetic acid (NAA) (I mg J"l) plus 6-benzylaminopurine (BA) (2 mg J·1) or kinetin (2 mg J·1) were the two hormone combinations added to the MS media. The explants were kept in constant high light intensity (33 llmol m·2 s·2 ) at 25°C. No contamination occurred. Fifteen percent of the leaf explants produced wound callus (Figure IB) but this soon browned and died. The peduncle explants also turned brown and died. One stolon of the plant was then used as explant source. The stolon was dipped in 70% ethanol for I min followed by 5 min in 0.2% Benlate and lastly, in full strength Jik (3.5% NaOCI) for 10 min. The stolon was then rinsed three times in sterile water and cut up and placed onto MS medium supplemented with the same
B.G. McAlister and J. van Staden* Natal University Research Unit for Plant Growth and Development, Department of Botany, University of Natal Pietermaritzburg, Private Bag X01, Scottsville, 3209 Republic of South Africa Received 23 January 1996; revised 27 April 1996 In vitro shoot formation was initiated us ing the apical regions of a stolon of Kniphofia pauciflora. Murashige and Skoog medium supplemented with 100 mg 1"1 myo-inositol, 3% sucrose a nd solidified with 0.8% agar was used. Kinetin (2 mg 1"1) and NAA (1 mg 1"1) were used to obtain shoot development. Shoots were subsequently placed onto hormone-free medium for rooting. Plantlets were successfully hardened off in a seedling mix in the mist house for 2 weeks, whereafter they were placed into a greenhouse and watered twice weekly.
*To whom correspondence should be addre.1sed.
S. Afr. J. Bot. 1996, 62(4)
220
hormonal treatments as the leaf and peduncle explants outlined above. There was a 60% contamination rate. No response was obtained 1 em below the apical region of the stolons using the two hormonal treatments. However, the upper I em of the apical
region of the stolon, which was cut into three ex plants, produced one or two shoots per explant. These shoots were excised and placed onto media containing 2 mg J·1 kinetin and I mg J-1 NAA. They produced additional shoots around their bases (Figure 1C).
•
D Figure 1 A, Kniphojia paucijlora Bak. B, Leaf explant with small amounts of wound callus on the cut surface. C, An older shoot (on the left) which has produced two new side shoots (middle and right) from the base of the older shoot. 0 , Production of roots on hormone-free medium.
S. Afr. J. Bot. 1996, 62(4): 221- 223
The maximum number of shoots produced from the base of an older shoot was three, but once the new shoots were excised, shoot production was more prolific. Some of the shoots were placed o nto hormone-free medi a to allow rooting to occur (Figure lD). The plants were then removed from culture and planted in a fine bark seedling mix. They were placed in a mist house with overhead misting and bottom heat (30°C) for 2 weeks to acclimatize. After this, the plants were placed in a greenhouse and watered twice week ly. There was an 80% survival rate. Although this is a fairly slow means of obtaining numerous plants, it is faster than vegetative propagation via the splitting of sto lons. As seeds are unobtainable, in vitro culture is an excellent means of conserving and increasing the numbers of this plant.
Acknowledgements The University of Natal Research Fund and the Foundation for Research Development are thanked for financial support.
References CODD, L.E. 1968. The South African species of Kniphojia. Botha/ia 9: 438-439. DAHLGREN. R.M.T.. CLIFFORD H.T. & YEO P.F. 1985. The families of the Monocotyledons: structure, evolution and taxonomy, pp. 179182. Springer-Verlag. Berlin. MURASHIGE, T. & SKOOG, F. 1962. A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Phy.fiologia Pl. 15: 473-497. NAYAK, S. & SEN, S. 1990. Differential requirement for organogenesis in two species of Kniphojia. Ind. J. exp. Bioi. 28: 171- 173.
221
the lateral resolution is limited by the wavelength of the light in use, typically to about 0.55 j.lm. Scanning electron microscopy (SEM) has a lateral resolution o f typically 5 nm. Both microscopes measure the size of features in the x and y directions (in the sample surface plane) but, however, neither provide any measurements in the z direction (normal to the sample surface plane). Measurement in the z direction requires cross-sectioning of the sample, which is quite a delicate technique. AFM, by contrast, while providing a lateral resolution of the same order of magnitude as SEM, allows direct measurement of the height of features with a resolution better than 0.1 nm. The exceptional three-dimensional imaging capability has, however, to be paid for by a limited vertical range, typically of a few microns. AFM has been very successfully used in the imaging of biological specimens (Radmacher et al. 1992). Because of the high spatial resolution and the unique vertical information allowed by AFM, we wanted to test the technique to image the fine ornamentation of a pollen grain. Aptenia cordfolia L.f., a member of the Aizoaceae fami ly, was chosen as a test sample to assess the ability of AFM to image the fine structure of a pollen grain. It presents a relatively smooth surface without spiny structures which would exceed the vertical range of AFM or interfere with the probe. It was also easily collected from outside our laboratory. The po llen grains were prepared by the acetolysis technique (Erdtman 1960). AFM, in its most basic form, scans a sample under a tip with a radius of between 10 and 40 nm at the e nd of a soft cantilever.
a
A 800 600
400
Atomic force microscopy images of a pollen grain: A preliminary study
200 0
C.M. Demanet* and K. Vijaya Sankar Department of Physics, University of Transkei, Private Bag X1, Umtata, 5100 Republic of South Africa Received II March 1996: revised 7 May 1996
We present in this short communication the firs t, to our knowledge, non-contact atomic force microscopy {AFM) image of a pollen grain. The advantages of non-contact AFM for the Imaging of pol· len grains reside in the absence of preparation required, the high spatial res olution, compared with S EM Images, and the unique three-dimensional rendition of the s urface topography, which avoids diffcult cross-sectioning of the sample. The ma in limitation is the restricted depth of field. AFM is therefore a valuable complementary microscopy technique for the study of the pollen grain's surface morphology.
b
A 1000500 0
'
Keywords: Non contact atomic force microscopy, pollen.
)Jm
)Jm
·ro whom correspondence should be addressed.
0 Optical and electron microscopy are routine tools in palynology. Recently, the introduction of atomic force microscopy (APM) (Binnig et at. 1986) has extended the range of techniques for the imaging of the morphology of surfaces. In optical microscopy
.0
Figure 1 Plane (a) and three-dimensional (b) representation of the surface of the pollen grain. The grey scale covers 100 nm in height. Note the difference in scale between lateral and vertical dimensions.
2I9
leaf anatomical evidence for regarding Chortolirion as a monotype, it is proposed that the genus should still be maintained separate from Haworthia. This stems primarily from its bulbous vegetative morphology and acuminate capsules, both features being absent in Haworrhia. Futhermore, it is the only haworthioid species of which the leaves are deciduous and die back to ground level after !ires or frost. It is, however, clear that the outer periclinal epidermal cell walls of Clwrtolirion are strongly PAS-positive, whereas, in H. blackbumiae and H. graminifolia, the corresponding walls are only slightly so (Figures 1-3), indicating a general lack of complex polysaccharides in the graminoid haworthias. A difference in fixatives could not have been a causal factor; the epidermal layers of H. kingiana and H. koe/maniorum showed PAS-positivity after treatment with the same fixative as the graminoid haworthias (compare Figures 5 & 6 with Figures 2 & 3). Although this featu re could be an additional diagnostic character for separating Chortolirion from grass-leaved species of Haworthia, a more comprehensive and detailed study is required before inferences can be made of its taxonomic value.
In vitro propagation of Kniphofia pauciflora Bak. for conservation purposes
Acknowledgements Mr Ian Oliver, curator of the Karoo National Botanical Garden, Worcester, is thanked for providing material of H. blackbumiae and H. graminifolia.
Keywords: Indigenous, Kniphofia pauclflora, micropropagation, rare, tissue culture.
References BAYER. M.B. 1972. Reinstatement of the genus A.mo/oba and Poe/1nitzia Uitw. (Liliaceae-Aioineae). Nat. Cact. Scux. J. 27: 77-79. COLLINS, B.A. & MACNICHOL. E.F. Uun.) 1978. Long term fixation for histological studies. Mkms,·. Al."ta 81: 155-158. FEDER. N. & O'BRIEN. T.P. 1968. Plant microtechnique: some principles and new methods. Am. J. Bot. 55: 123- 142. HILTON-TAY LOR. C. & SMITH. G.F. 1994. The conservation status of Aloaccae 111 southern Africa. In: Botanical diversity in southern Africa, cd. B.J. Huntley. pp. 287-303. Stre/iczia l. National Botanical Institute, Prctona. MUELLER. W.C. & GREENWOOD. A.D. 1977. The ultrastructure of phenolic cells lixed with caffeine. J. exp. Bot. 29: 757-764. SMITH. G.F. 1990. Nomenclatural notes on the subsection Bowieae in Aloe (Asphodeluceae: Alooideae). S. Afr. J. Bot. 56: 303-308. SM ITH. G.F. 199 1. Studies on the reproductive biology and palynology of Clwrtoliritm Berger (Asphodelaccae: Aloaceae) in southern Africn. Taxon40 : 61-73. SMITH. G.F. 1994. Taxonomic history of Poellnitzia Uitcwaal, a unispccific genus of Alooideae (Asphodelaceae). Haseltonia 2: 74-78. SMITH. G.F. & VAN WYK. A.E. 1992. Systematic leaf anatomy of selected genera of southern African Alooideae (Asphodelaceae). S. A.(1: 1. Bot. 58: 349- 357.543. SMITH. G.F. & VANWYK. A.E. 1993. The generic status of Chortolirion (Aioaccnc). Kt•w Bull. 48: lOS-I 13. SM ITH. G.F. & VAN WYK. B-E. I991. Generic relationships in the Alooideac (Asphodelacene). Taxon 40: 557-581.
Kniphofia paucijlora Bak. is a pretty plant belonging to the subfamily Asphodeloideae (Dahlgren et al. 1985). The plants have short stolons, forming groups of short stems. The 20-35-cm leaves are soft and yellow green. The leaves are 2- 8 mm wide and are triangular in cross section (Codd 1968). The flower spike is 30-50 em long and is raised above the leaves. The inflorescence is yellow and few-flowered compared to other Kniphojia sp. (Figure I A). These plants flower from September to November. Kniphojia paucijlora is known only from restricted areas near Durban in Natal, where it used to grow in marshy grasslands. This species is threatened due to urbanization and is no longer found at the type locality. Specimens were found in the Marian Hill area in 1951 and I 954 but the habitat has since been drained and the plants have disappeared from this region (Codd 1968). Conservation of this plant is needed and tissue culture was undertaken to increase the limited numbers held in the National Botanical Garden in Pietermaritzburg. Only one reference to the in vitro culture of Kniphofia species could be found (Nayak & Sen I 990). A single plant was obtained from the Botanical Garden in Pietermaritzburg to use as explant source. First attempts at culturing this plant were undertaken using leaf and peduncle explants. The material was sterilized for 5 and 10 min using 3.5% sodium hypochlorite and then placed on Murashige and Skoog (1962) medium (MS) solidified with 0.8% Unilab agar. The pH was adjusted to 5.8 with I M NaOH prior to being autoclaved. 1-Naphthaieneacetic acid (NAA) (I mg J"l) plus 6-benzylaminopurine (BA) (2 mg J·1) or kinetin (2 mg J·1) were the two hormone combinations added to the MS media. The explants were kept in constant high light intensity (33 llmol m·2 s·2 ) at 25°C. No contamination occurred. Fifteen percent of the leaf explants produced wound callus (Figure IB) but this soon browned and died. The peduncle explants also turned brown and died. One stolon of the plant was then used as explant source. The stolon was dipped in 70% ethanol for I min followed by 5 min in 0.2% Benlate and lastly, in full strength Jik (3.5% NaOCI) for 10 min. The stolon was then rinsed three times in sterile water and cut up and placed onto MS medium supplemented with the same
B.G. McAlister and J. van Staden* Natal University Research Unit for Plant Growth and Development, Department of Botany, University of Natal Pietermaritzburg, Private Bag X01, Scottsville, 3209 Republic of South Africa Received 23 January 1996; revised 27 April 1996 In vitro shoot formation was initiated us ing the apical regions of a stolon of Kniphofia pauciflora. Murashige and Skoog medium supplemented with 100 mg 1"1 myo-inositol, 3% sucrose a nd solidified with 0.8% agar was used. Kinetin (2 mg 1"1) and NAA (1 mg 1"1) were used to obtain shoot development. Shoots were subsequently placed onto hormone-free medium for rooting. Plantlets were successfully hardened off in a seedling mix in the mist house for 2 weeks, whereafter they were placed into a greenhouse and watered twice weekly.
*To whom correspondence should be addre.1sed.
S. Afr. J. Bot. 1996, 62(4)
220
hormonal treatments as the leaf and peduncle explants outlined above. There was a 60% contamination rate. No response was obtained 1 em below the apical region of the stolons using the two hormonal treatments. However, the upper I em of the apical
region of the stolon, which was cut into three ex plants, produced one or two shoots per explant. These shoots were excised and placed onto media containing 2 mg J·1 kinetin and I mg J-1 NAA. They produced additional shoots around their bases (Figure 1C).
•
D Figure 1 A, Kniphojia paucijlora Bak. B, Leaf explant with small amounts of wound callus on the cut surface. C, An older shoot (on the left) which has produced two new side shoots (middle and right) from the base of the older shoot. 0 , Production of roots on hormone-free medium.
S. Afr. J. Bot. 1996, 62(4): 221- 223
The maximum number of shoots produced from the base of an older shoot was three, but once the new shoots were excised, shoot production was more prolific. Some of the shoots were placed o nto hormone-free medi a to allow rooting to occur (Figure lD). The plants were then removed from culture and planted in a fine bark seedling mix. They were placed in a mist house with overhead misting and bottom heat (30°C) for 2 weeks to acclimatize. After this, the plants were placed in a greenhouse and watered twice week ly. There was an 80% survival rate. Although this is a fairly slow means of obtaining numerous plants, it is faster than vegetative propagation via the splitting of sto lons. As seeds are unobtainable, in vitro culture is an excellent means of conserving and increasing the numbers of this plant.
Acknowledgements The University of Natal Research Fund and the Foundation for Research Development are thanked for financial support.
References CODD, L.E. 1968. The South African species of Kniphojia. Botha/ia 9: 438-439. DAHLGREN. R.M.T.. CLIFFORD H.T. & YEO P.F. 1985. The families of the Monocotyledons: structure, evolution and taxonomy, pp. 179182. Springer-Verlag. Berlin. MURASHIGE, T. & SKOOG, F. 1962. A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Phy.fiologia Pl. 15: 473-497. NAYAK, S. & SEN, S. 1990. Differential requirement for organogenesis in two species of Kniphojia. Ind. J. exp. Bioi. 28: 171- 173.
221
the lateral resolution is limited by the wavelength of the light in use, typically to about 0.55 j.lm. Scanning electron microscopy (SEM) has a lateral resolution o f typically 5 nm. Both microscopes measure the size of features in the x and y directions (in the sample surface plane) but, however, neither provide any measurements in the z direction (normal to the sample surface plane). Measurement in the z direction requires cross-sectioning of the sample, which is quite a delicate technique. AFM, by contrast, while providing a lateral resolution of the same order of magnitude as SEM, allows direct measurement of the height of features with a resolution better than 0.1 nm. The exceptional three-dimensional imaging capability has, however, to be paid for by a limited vertical range, typically of a few microns. AFM has been very successfully used in the imaging of biological specimens (Radmacher et al. 1992). Because of the high spatial resolution and the unique vertical information allowed by AFM, we wanted to test the technique to image the fine ornamentation of a pollen grain. Aptenia cordfolia L.f., a member of the Aizoaceae fami ly, was chosen as a test sample to assess the ability of AFM to image the fine structure of a pollen grain. It presents a relatively smooth surface without spiny structures which would exceed the vertical range of AFM or interfere with the probe. It was also easily collected from outside our laboratory. The po llen grains were prepared by the acetolysis technique (Erdtman 1960). AFM, in its most basic form, scans a sample under a tip with a radius of between 10 and 40 nm at the e nd of a soft cantilever.
a
A 800 600
400
Atomic force microscopy images of a pollen grain: A preliminary study
200 0
C.M. Demanet* and K. Vijaya Sankar Department of Physics, University of Transkei, Private Bag X1, Umtata, 5100 Republic of South Africa Received II March 1996: revised 7 May 1996
We present in this short communication the firs t, to our knowledge, non-contact atomic force microscopy {AFM) image of a pollen grain. The advantages of non-contact AFM for the Imaging of pol· len grains reside in the absence of preparation required, the high spatial res olution, compared with S EM Images, and the unique three-dimensional rendition of the s urface topography, which avoids diffcult cross-sectioning of the sample. The ma in limitation is the restricted depth of field. AFM is therefore a valuable complementary microscopy technique for the study of the pollen grain's surface morphology.
b
A 1000500 0
'
Keywords: Non contact atomic force microscopy, pollen.
)Jm
)Jm
·ro whom correspondence should be addressed.
0 Optical and electron microscopy are routine tools in palynology. Recently, the introduction of atomic force microscopy (APM) (Binnig et at. 1986) has extended the range of techniques for the imaging of the morphology of surfaces. In optical microscopy
.0
Figure 1 Plane (a) and three-dimensional (b) representation of the surface of the pollen grain. The grey scale covers 100 nm in height. Note the difference in scale between lateral and vertical dimensions.