Amer J of Potato Res (1999) 76:9-16
9
Screening Diploid S o l a n u m Genotypes Responsive to Different Anther Culture Conditions and Ploidy Assessment of Anther-Derived Roots and Plantlets A. N a s e e r Aziz 1, J a n e t E. A. S e a b r o o k 2., G e o r g e C. C. Tai 2, a n d Hielke De J o n g 2 ~Department of Biology,Universityof New Brunswick, Bag ServiceNo. 45111, Fredericton, N. B., Canada E3B 6El. E-mail:
[email protected]. 2potato Research Centre, Agriculture& Agri-FoodCanada, P.O. Box 20280, Fredericton, N. B., Canada E3B 4Z7. E-mail:
[email protected],
[email protected],
[email protected]~ *Correspondingauthor phone number: (506)452-3260.
ABSTRACT
INTRODUCTION
T w e n t y - t h r e e diploid ( 2 n = 2 x = 2 4 ) p o t a t o c l o n e s w e r e screened for r e s p o n s e to four different anther culture media. Anther derived tissues (calll/embryos) were obtained from s e v e n o f the clones in liquid culture media. Anther culture r e s p o n s e o f t h e s e s e v e n clones was further quantified on five additional media. Since clonal r e s p o n s e s are specific to culture conditions (Tiainen, 1992), the seven genotypes s o m e w h a t d i f f e r e d f r o m e a c h o t h e r in the m a g n i t u d e o f t h e r e s p o n s e t o e a c h o f t h e media. In a g r e e m e n t w i t h Heberle-Bors (1985 ) a more realistic assessment o f the r e s p o n s i v e n e s s was possible when pollen counts per anther were used to express the yield o f anther-derived material. A relatively higher level of exogenous c y t o k i n i n ( 6 - b e n z y l a d e n i n e ) to that o f a u x i n (3-indoleacetic acid) proved to be favourable for the inducton o f calli/embryos. Use o f a semi-solid medium saved s p a c e and was c o n v e n i e n t for culture o b s e r v a t i o n s therefore it was found more conducive for the genotypic screening. Anther derived tissues that regenerated to produce root tissue and/or plantlets were f u r t h e r multiplied o n a r o o t culture m e d i u m and a plantlet medium. Liquid culture conditions on orbital shakers were conducive for the growth of root explants. Anther derived material ( r o o t s and p l a n t l e t s ) origin a t i n g from c l o n e 9 5 0 7 - 0 4 r e v e a l e d d i f f e r e n c e s in growth vigor. Flow cytometric estimation o f ploidy in regenerated roots and plantlets, revealed that clone 9507-04 produced 44% monoploid roots and 77% monoploid plantlets. Seventy-three percent anther-derived plantlets from clone 6028-02 were monoploids.
Commercial potato cultivars are heterozygous tetraploids (2n=4x=48) and F 1 progenies are genetically highly variable (Uijtewaal eta/., 1987). Dihaploid (2n=2x=24) plants can be produced from commercial cultivars by anther culture (CaUeberg, 1996) or prickle pollination (Hougas et al., 1964). Monoploids (2n=1x=12) can similarly be produced from dihaploids or diploid species (Uijtewaal et al., 1987 and Calleberg, 1996). Anther culture of diploid potatoes to produce monoploid plants and recurrent selection for regenerability can counter-select lethal and/or sublethal alleles (Meyer et al., 1993). Homozygous doubled monoploid clones derived from anther culture can be used in breeding as well as genetic studies (Veilleux eta/., 1995; Yamagishi et al., 1996). Potato genotypes differ in their potential for embryogenesis and regeneration under anther culture conditions (Uhrig, 1985). Therefore ff homozygons plants are to be regenerated via androgenesis, potato germplasm has to be screened for anther culture response. The purpose of this study was to screen selected diploid Solanum clones for their regeneration response in anther culture. The potato clones were evaluated for the ability to regenerate anther-derived roots and/or plantlets using several different media. Flow cytometric estimation of the ploidy was used to assess the frequency of monoploid anther-derived roots and plantlets.
Acceptedfor publication June 16, 1998. ADDITIONALKEY WORDS:Androgenesis, flow cytometry, pollen, potato, root culture, tissue culture.
MATERIALS AND METHODS Greenhouse Stock Plants--Field grown diploid potato clones (Table 1) were maintained at the Benton Ridge Potato Breeding Substation of the Potato Research Centre of Agriculture and Agri-Food Canada: Tubers were potted in 2 L peat pots containing composted soil. Plants were grown in a greenhouse and fertilized every 2 weeks (600 ppm N2 per application) with soluble 20% N: 8.7% P: 16.6% K fertilizer
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AMERICAN JOURNAL OF POTATO RESEARCH
Vol. 76
(Plant Products Co. Ltd.), and watered as required. In winter
was also estimated by using a haemocytometer. Unopened
m o n t h s the natural day length was supplemented with 1:3
flower buds were harvested and placed in plastic vials ( - 5
c o m b i n a t i o n of i n c a n d e s c e n t and fluorescent lamps (cool
ml) with 1-2 m m diameter holes p u n c h e d on the sides. The
white F96T12/cw) for a total light intensity of 1000 ]JEm-2s-1
vials were wrapped in moist paper-towels and cold treated
to provide 16 h photoperiod. Greenhouse temperature was
at 4-5 C in a refrigerator for 72 h. Flower buds were surface-
maintained between 20+7 C (night) and 26+7 C (day). High
sterilized by immersion in 70% ethanol for 30 seconds fol-
humidity maintained by wetting the greenhouse floor using
lowed by 15 m i n u t e s in 10% Javex-12 (commercial bleach
sprinklers (5 minutes) of water at 30 minute intervals was used to reduce flower abscission. Stem cuttings (Seabrook,
containing 10.85% (w/w) sodium hypochlorite) and rinsed
1990) were also used to propagate plants. The rooted cut-
twice in sterilized distilled water. The buds were dissected and the anthers excised.
tings were then grown in the abovementioned flower promoting conditions in the greenhouse.
ture media (Table 2). Four different media were used in the
f l o w e r B u d Selection a n d T r e a t m e n t - - T h e lengths of
flower b u d s from selected clones were correlated to the uninucleate or early binucleate microspore developmental stages (Table 1). Acetocarmine (4% carmine in 45% acetic acid) was applied to stain the nuclei in microspores at successive developmental stages. For each clone approximate counts of pollen per anther (average of at least two repeats)
A n t h e r C u l t u r e - - E x c i s e d anthers were placed in/on cul-
genotypic screening experiment and an additional semi-solid medium was used in the post-screening experiment (Table 2). For each clone at least three replicates were cultured: liquid media, three flasks containing 30 anthers each; semi-solid mediunl, three petri-dishes containing 20 anthers each. The excised anthers were placed into liquid media (15 ml aliquots) Uhr85, MSU93 and N=B96 (Table 2) in 50 ml
TABLE 1 . - - C o m p a r a t i v e traits o f 2 3 selected diploid potato clones used i n a n t h e r culture studies.
Clone
Background pedigree~
Flower bud (ram) length2
W5295.7 BPH32-03 8664-06 58794)6 9507-04 11364-03 113644)9 11364-22 11364-37 1136448 91204)5 19114)2 1911-19 1911-33 1911-48 11406-15 1140646 9465-03 9459-07 75064)1 6028-02 F20.1D F'20.lI
50% tb~, 50°/0phu/stn 50% tb~, 50%phu 50% tbr, 500/ophu/stn 50% tbr, 50°/0phu/stn 50°/0 tbr, 50°/0wild (chc, A~y.) 50o/0tbr,25°/0phu/stn,25°/~hc 50o/0tbr,25%phu/stn,25%chc 50o/otbr,25O/0phu/stn,25o/0chc 50o/0tbr,25%phu/stn,25%chc 50o/0tbr,25%phu/stn,25%chc 50°/0 tbr, 50%phu/stn 50% tb~, 50% phu/stn 50% tb~, 50% phu/stn 50°/0 tbr, 50% phu/stn 50°/0 tbr, 50°/0phu/stn 50% tbr, 50o/0phu/stn 50°/0 tbr, 50o/0phu/stn 50°/0 tbr, 50°/0phu/stn 50°/0 tbr, 50o/0phu/stn 50°/0 tb~, 50o/~phu 100% tbr 100°/o tbr 100°/0 tbr
5-7 8-9 7-8 7-8 5-6 9-10 64 5-7 8-9 64 5-7 6-8 8-9 6-8 7-8 7-8 8-9 7-8 9-10 7-9 8-9 9-10 9-10
Anther (mm) length2 2-3 3-4 3 3-4 3 3 3 2-3 3 34 3 3 34 2.5-3.5 3 2.5 2.5-3.0 3 34 2.5-3.0 2.5-3.0 3.0-3.5 3.0-3.5
Pollen (xl000) anther
Totalanthers cultured
Percent tissue yield3
100 120 30 140 175 50 95 90 140 130 55 115 40 125 100 130 140 240 250 270 105 80 125
450 1050 420 450 1230 360 1270 390 390 420 360 360 420 360 390 360 360 360 390 1070 670 850 660 Total= 13,040
0 9 (8X10-7) AS 0 104 (59X10-7) 0 18 (19X10-7) 0 AS AS 0 AS AS AS AS RS 0 AS RS 12 (5X10-7) 45 (42X10-7) 10 (13X10-7) 39 (31X10-7)
ltbr= S. tuberosum, phu= S. phu~vja, stn= S. stenotomum, ehe= S. chacoense, Arg. =Argentinianwild potato species. 2. Containingmicrospores at unlnucleate or early binucleate stage, 3. Anther-derivedtissue yield per 100 anthers cultured and figures in parentheses represent anther-derived tissue yield per 100 microspores cultured. AS= Response shown by only anther swelling;RS= Only rare swelling of the anthem was observed.
AZIZ, eta/.: CULTURE OF ANTHERS
1999
11
TABLE 2.---Composition of seven media used f o r diploid potato anther culture.
Constituents
MS basal salts6 (g/L) Sucrose (g/L) Charcoal (g/L) Wheat starch (g/L) Agar (~JL) Inositol (mg/L) Thiamine.HC1 (mg/L) Ascorbic acid (mg/L) Glutamine (mg/L) Asparagine (mg/L) Cystein.HC1 (mg/L) Lysine.HC1(mg/L) IAA(mg/L) NAA (mg/L) BA (mg/L) GA3 (mg/L)
Uhr851
MSU932
S&V953
RVP954
N=B965
Morphogenic mediumI
Plantlet mediumI
2.16 60 0.5 0 0 50 0.2 0 0 0 0 0 0.1 0 2.5 0
2.16 60 0.5 0 0 50 0.2 0 7.3 6.6 O 0 0.1 0 1 0
2.16 60 2.5 0 0 100 0.4 0 0 0 0 0 0.1 O 2.5 0
4.31 60 5 30 0 0 0 200 0 0 60 10 0 0 3 0
2.16 60 0.5 0 0 50 0.2 0 0 O 0 0 0 1.5 1.5 0
2.16 20 O 0 8 50 0.2 0 0 0 0 0 0 0 0 0.1
2.16 20 O 0 8 50 0.2 0 0 0 0 0 0 0 0 0
1Uhrig, 1985. 2Meyer eta/, 1993;. 3Shen and Veilleux, 1995a. 4Rokka et al, 1995. 5Medium containing equal concentration of NAA and BA. 6Murashige and Skoog, 1962. Erlenmeyer flasks which were c a p p e d with n o n - a b s o r b e n t cotton and w r a p p e d with almunium foil. Cultures were incub a t e d at either 20 lJEm-2s -1 light intensity, ambient temperat u r e a n d d a y l e n g t h in t h e s c r e e n i n g e x p e r i m e n t , o r 1 ]JEm-2s -1 light i n t e n s i t y (Uhrig, 1985), 25 C a n d 16 h d a y length (Meyer et al., 1993) in the post-screening experiment. S&V95 (Table 2) was distributed in 125 ml Delong culture flasks and cultures were maintained as described (Shen and Veilleux, 1995a). All liquid cultures were inoculated with cold treated anthers and were maintained at 120 rpm (Uhrig, 1985; Shen and Veilleux, 1995a) shakers. RVP95 (Table 2) m e d i u m (20 ml) was distributed in 90 m m (diameter) sterile plastic petri dishes. Fresh or cold incubated anthem were p l a c e d on the m e d i u m and the petri dishes were sealed with parafilm. The cultures were maintained at 28 C, with light intensity of 45 llEm-2s-1 (cool-white, F20T12/CW, fluorescent lamps) and 16 h day length (Rokka e t a / . , 1995). All media ( p H 5.8+0.1) were autoclaved before dispensing.
Culture of Anther-Derived Tissues--Anther-derived embryos and/or calluses were harvested from 5 to 12 w e e k o l d c u l t u r e s in liquid m e d i a a n d s u b c u l t u r e d on t h e morphogenic medium (Uhrig, 1985). After 4-8 w e e k s on the morphogenic medium, the anther-derived tissue developed into r o o t s or plantlets. Root tissues were harvested and subcul-
tured on liquid and semi-solid (0.8% agar) White's m e d i u m (White, 1963) containing 2% sucrose a n d 5.8+0.1 pH. Semisolid m e d i u m was dispensed in 60 m m (diameter) single-weU petri dishes. Liquid medium was dispensed either in 6-well plates or as 15 ml aliquots in 50 ml Erlenmeyer flasks. Root cultures were maintained at 19+1 C and continuous darkness for 16-18 weeks. The liquid cultures in Erlenmeyer flasks were placed on 120 r p m shakers. Plantlets developed on morphogenic m e d i u m were cultured on the plantlet m e d i u m (Uhrig, 1985). Anthers on the semi-solid m e d i u m (Rokka eta/., 1995) yielded plantlets in 2-5 months. These plantlets were excised and subcultured on a plantlet m e d i u m (Uhrig, 1985). Flow C y t o m e t r y - - S a m p l e s w e r e p r e p a r e d f o r f l o w cytometry following the p r o c e d u r e of Martin and Widholm (1996). J u s t b e f o r e a n a l y s i s on a C o u l t e r EPICS-XL flow c y t o m e t e r (Dr. Everett Chalmers Hospital, Fredericton, N. B.) the s a m p l e s were p a s s e d through 52 Inn mesh (Spectra/ Mesh, Spectrum) into 3 ml p o l y s t y r e n e tubes. Single param e t e r histograms were gated on FALS (forward angle light scatter) vs. 90 ° LS (light scatter) for size measurements and i n t e g r a t e d RFL ( r e d f l u o r e s c e n c e ) vs. p e a k RFL for DNA m e a s u r e m e n t s . MULTICYCLE DNA a n a l y s i s s o f t w a r e (Phoenix F l o w Systems) was u s e d for d a t a collection and analysis (2,000-5,000 nuclei p e r sample). Peaks were scored
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AMERICAN JOURNAL OF POTATO RESEARCH
as diploid or monoploid compared to known diploid leaf samples (anther donor) used as standards in each run of the flow cytometer. At least six samples were analyzed in two or more separate runs of the flow cytometer to confirm the results.
RESULTS AND DISCUSSION Screening Potato Genotypes f o r A n t h e r Culture Response--Genotypic differences were observed for the relationship between flower bud length (5 to 10 ram) and anther length (2 to 4 mm) corresponding to the uninucleate/early binucleate stage of m i c r o s p o r e d e v e l o p m e n t (Table 1). Pollen prodution of clones also varied markedly (30,000 to 270,000) per anther (Table 1). There was no apparent clonal relationship between pollen production per anther and regenerability (Table 1). All 23 clones were cultured in four different liquid media (Table 2). Only seven clones (BPH32-03, 95074)4, 113644)9, 7506-01, 6028-02, F20.1D & F20.1I) were responsive to the anther culture conditions in the screening experiment. Anther-derived tissues (calli and/or embryos) sloughed off from anthers in the liquid media. Total yield of anther-derived tissue (calli and/or embryos) in the liquid culture media was used as the anther culture response-index of clones and is given in Table 1. These results confirmed previous reports (Rokka et al., 1995; Uhrig, 1985) that genotypic control is the most important factor in anther culture response. The results agreed with previous findings that clones W5295.7, 8664-06 and 5879-06 were unresponsive, and clone 6028-02 was responsive to the anther culture (Janjua, 1988). Clones F20.1D and F20.1I are anther-derived progeny of the clone 6028-02 (Cappadocia-personal communication; Janjua, 1988). Apparently both clones retained the culture responsive quality of their donor (6028-02). Since several Solanum chacoense derived clones were reported to be responsive to anther culture (Rivard et al., 1996) the S.chacoense background in clone 9507-04 may also have been contributory to its success in anther culture. The response of clone 7506-01 to anther culture conditions was low (12%). However, Mozafari et al. (1995) reported regeneration from leaf discs and stem internodes of this clone but did not state regeneration frequency. Genotypic Variation in Response to Culture M e d i a - Anthers from seven responsive clones were cultured on five different culture media in the post-screening experiment. All clones had more or less different response to each medium (Table 3). Exogenous cytokinin (6-benzyladenine) was com-
Vol. 76
mon in all four media used in the experiments. Equal concentrations of exogenous auxins (1-naphthylacetic acid) and BA used in the medium N=B96 (Table 2) did not induce maximal response except in clone F20.1D (Table 3) confirming the reports of Rihov~, and Tup~ (1996) and Tiainen (1993). Medium S&V95 (Table 2) was conducive for anther-derived tissue yield from clones BPH32-03, 9507-04, 11364-09 and 7506-01 (Table 3). Clones 6028-02, F20.1D and F20.1I had more tissue yield respectively on media Uhr85, MSU93 and N=B96 c o m p a r e d to other media used. Clone 6028-02 appeared to prefer the minimal culture conditions of Uhr85 medium (Table 2), whereas the amino acid supplements in MSU93 (Table 2) were favourable for clone F20.1I. Specific genotypes have been reported to respond differently on various culture media (Tiainen, 1992). The yield of anther-derived tissue per 100 anthers cultured from the clones in liquid media was as follows; 950704, 104%; 6028-02, 45%; F20.1I, 39%; 11364-09, 18%; 7506-01, 12%; F20.1D, 10% and BPH32-03, 9%. When pollen per anther was used the above ranking was modified, so that clone F20.1D appeared to be more responsive to culture conditions followed by BPH32-03 and 7506-01 (Table 1). We therefore recommend, in agreement with Heberle-Bors (1985), that for a more realistic assessment of the anther-derived tissue yield, pollen grains rather than anthers should be stated as units. Semi-Solid M e d i u m and Cold Pre-Treatment of Anthers--Anthers without cold treatment were cultured on semi-solid RVP95 medium (Table 2) which allowed the simultaneous screening of all genotypes in a limited area. Semi-solid m e d i u m allowed convenient observation of anthers undergoing culture induced morphological changes without disturbing the culture. Cold pre-treated anthers of clones 9507-04 and 602802 were cultured on medium RVP95; 34% of the cold treated anthers from clone 9507-04 produced calli/embryos compared to 16% of untreated anthers (Table 3). Cold pre-incubation also reduced time required to initiate culture response from 7 to 5 weeks (Table 3). Clone 6028-02 exhibited reduction in culture response of cold treated anthers (8%) compared to untreated anthers (17%). Five weeks were required to initiate calli/embryo production from clone 60284)2 with or without 4 C pre-incubation of the anthers. Anther-Derived Roots and Plantlets--Anther-derived tissues (calli and embryos) from each clone were subcultured on the morphogenic medium (Table 2) to produce roots and/or plantlets. On the morphogenic medium some anther-
1999
AZIZ, et ed.: CULTURE OF ANTHERS
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TABLE 3.--Relative anther culture response of seven diploid potato clones. Percent tissue yield (%) per number of anthers cultured and time (weeks) to initiate anther culture response in liquid media Potato Clones
Uhr851 %Response weeks
MSU932 %Response weeks
S&V953 %Response weeks
%regenerating anthers
N=B964 %Response weeks
BPH32-03 9507-04
8 29
5 7
1 48
12 7
28 262
6 7
0 78
9 6
11364-09 7506-01 6028-02
3 8 95
10 5 4
23 10 27
10 5 4
45 26 51
12 8 6
0 4 6
10 5 7
1
12 10
10 137
8
14
7
11
10 5
19 5
11 5
F20.1D F20.1I
4
RVP95t %Response weeks N39 N16 C34 N53 N18 N17 C08 N20 N18
5 75 5 12 55 7
5
1Uhrig, 1985. 2Meyeret al, 1993. 3Shen and Veilleux, 1995a. 4Medium containingequal concentrationsof NAAand BA. tPercent anthers respondingper numbers of anthers cultured, on Rokkaet al (1995)semi-solidmedium, with (C) or without (N) cold l~eatment.
derived tissues did not survive and others proliferated only to callus. Roots were regenerated from all seven responsive clones. Thirty-four regenerated roots from clone 9507-04 were subcultured on root culture medium (White, 1963). The roots proliferated in liquid medium with agitation but not in stationary medium. Thirteen cultured roots did not grow on any medium whereas 10 cultured roots grew only on liquid medium. Eleven roots showed growth on both liquid and semi-solid media however there was comparatively less root growth on semi-solid medium. Plantlets were regenerated only from clones 9507-04 and 6028-02, perhaps because of the greater number of embryos produced by them. Since only a few embryos are apparently capable of regenerating into plantlets (Shen and Veilleux, 1995b), a higher embryo yield would increase the chance of obtaining anther-derived-plantlets from a particular genotype. Conversion rates from embryo to plantlet is also under genetic control (Shen and Veilleux, 1995b). Clones producing large numbers of embyros that did not convert efficiently into plantlets have been reported (Meyer et al., 1993). Clone 9507-04 produced a large mass of anther derived tissue (callYembryos) and plantlets later emerged from these structures (Figure 1, a) whereas clone 6028-02 had a tendency to produce plantlets directly emerging from the anthers (Figure 1, b). This difference was probably reflective of the genotypic dependence of the sporophytic pollen development either
through pollen callogenesis or by direct embryogenesis (Rihovfi, and Tup~, 1996). Regenerated plantlets were subcultured on growth regulator-free semi-solid medium (Table 2). Differences in growth habits of regenerants from a single clone were observed as 4 roots from regenerated tissues of the clone 9507-04 were able to grow faster on both liquid and semi-solid media as eompared to other 6 whereas 10/36 roots grew on liquid medium only. Similar differences in growth habit and structural features (pigmentation and size of leaves, etc.) were also observed for the anther-derived plantlets. DNA content (ploidy) of anther-derived plantlets (from clones 9507-04 and 6028-02) and root tissues (from clone 9507-04) was ~ a t e d by flow cytometry (Figure 2). Antherderived material from diploid plants was identified as either containing DNA content of a monoploid (lx) or higher (2x) ploidy (Figure 2). Diploid potato can produce diploid plants from anther culture through unreduced gametes or by spontaneous chromosome doubling during or after culture (Veilleux eta/., 1995) and this phenomenon is also influenced by the genotype (Pijnacker eta/., 1989). Flow cytometric estimation of the initial regenerants showed that clones 9507-04 and 6028-02 produced 77% and 73% monoploid anther-derived plantlets, respectively. Monoploid regenerants were less frequent in anther-derived root tissues as only 44% of roots, regenerated from the clone 9507-04, were found to be monoploid.
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AMERICAN JOURNAL OF POTATO RESEARCH
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FIGURE 1 Regenerating anther-derived plantlets. (a): Plantlets emerging from callus of clone 950704, Xl.7; (b): Developed anther-derived embryos from clone 6028-02, X4.0.
In this report five anther culture media were evaluated for efficient response of diploid potato material and in agreement with Heberle-Bors (1985), an accurate comparative assessment of the responsiveness was possible when pollen counts per anther were used. A semi-solid medium was convenient for simultaneous screening of several genotypes using only limited culture space. Genetic markers associated with regeneration during anther culture (Xu et al., 1997) could be developed to identify and preselect culture responsive potato clones. Subsequent yield of anther-derived material from pre-screened responsive clones was efficient on liquid media which contained higher BA concentration com-
pared to IAA. Genotypic variations were noted in the degree of anther culture response, the ability to regenerate either or both roots and plants, and the mode of regenerating plants either direct from anther or via anther-derived tissue. Monoploid anther-derived roots and plantlets can be used for genetic studies. Here we report for the first time the ploidy assessment of roots derived from anther culture of a potato clone. We suggest a similar use of anther-derived roots in genetic segregation analysis as reported for anther-derived potato plants (Veilleux et al., 1995). RAPD based genetic analysis of monoploid roots and plants derived from anthers of clone 9507-04 is currently in progress in our laboratory.
1999
AZIZ, et a/.: C U L T U R E O F A N T H E R S
15
f o r o p e r a t i n g t h e f l o w c y t o m e t e r . R e v i e w o f Materials a n d ix
M e t h o d s s e c t i o n b y L. K a t h e r y n D o u g l a s a n d Coral Curtis is
°
2X
also acknowledged. Department
This research
was supported
by
of Biology, University of New Brunswick
t h r o u g h p r o v i d i n g a g r a d u a t e assistantship, and A g r i c u l t u r e a n d A g r i - F o o d C a n a d a b y p r o v i d i n g t h e r e s e a r c h facilities and support.
(a)
. . . . . . . . FL3
~O2'i
ot
. . . . . . . .
iaz,~
LITERATURE C I T E D
(b)
IX
_, , .
,,~ ,-~-7.t , I . ~3
(c)
. , 1024
I
8 . . . . . . . . Ft'l
102,J
(d) m~l ix
2X
i ~ ! ' ~ ' " 'FL3 "' (e)
"~m
ol
. . . . FL3
10z~
FIGURE 2 E s t i m a t i o n o f t h e D N A c o n t e n t ( r e l a t i v e f l u o r e s c e n c e o f propidi u m i o d i d e - D N A s t a i n ) by f l o w c y t o m e t r y . X-axis r e p r e s e n t s t h e c h a n n e l n u m b e r ( 0-1024 ) a n d Y-axis gives t h e f r e q u e n c y o f nuclei in e a c h chamml. D N A p e a k s o n X-a~i~ o b s e r v e d ca. 120 for m o n o p l o i d ( l x ) a n d ca. 2 6 0 f o r d i p l o i d ( 2 x ) m a t e r i a l . ( a ) D i p l o i d a n t h e r d o n o r 9507-04, ( b ) M o n o p l o i d anther-derived r o o t t i s s u e from 9507-04, ( c ) Diploid a n t h e r - d e r i v e d p l a n t l e t from 9507-04, ( d ) M o n o p l o i d anther-derived p l a n t l e t from 9507-04, ( e ) Diploid anther donor 6028-02, (f) Monoploid anther-derived plantlet from 6028-02.
ACKNOWLEDGMENTS We thank Andrew Gardner and Steven Allaby for g r e e n h o u s e m a i n t e n a n c e o f t h e plants. The a u t h o r s t h a n k L. Katheryn Douglas for technical suggestions and Roger S m i t h for p h o t o g r a p h y . T h a n k s are also due to C o r a l Curtis
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