Indian Journal of Experimental Biology Vol. 38, June 2000, pp. 593-597

Microspore culture in Corchorus olitorius: Effect of growth regulators, temperature and sucrose on callus formation*

M Abbas Ali .. & 1 K Jones

Department of Agricultural Botany, University of Reading, Reading, England, U.K.

Received 12 October / 998; revised 9 March 2000

Culture of isolated microspores and of anthers on media containing IAA directed free microspore development to an embryogenic pathway in C. olitorius. The first division of microspores on transfer to culture media was symmetrical in contrast to the asymmetrical division seen in normal development in vivo. Initially, I 0-30% microspores divided symmetrically, but only 0.2- I% of the dividing microspores continued dividing and produced multicellular microcalli. About 30% of these microcalli produced callus but only on medium with 2.0 mg/L zeatin and 0.1 mg/L IAA. Incubation in the dark at temperatures of 35" C for I day and then 25" C was found effective for induction of first embryonic division in Corchorus. The frequency of microspore cal lus formation was higher on medium contai ning ei ther 3% or 5% sucrose. Addition of colchicine and addition of activated charcoal to the above medium did not enhance microspore division in Corchorus olitorius . On transfer to different media most calli produced roots but regeneration of shoots and embryos was not induced .

Anther culture and isolated microspore culture are now used in plant breeding for the production of haploid plants by inducing the development of uninucleate haploid microspores into plantlets. Haploid plants contain e ither a single set of chromosomes or the gametic number of chromosome sets, and the diploids or natural polyploids can be produced by doubling the chromosome complement of the haploids. In this way either anther culture or rnicrospore culture is used to produce genetically homozygous plants. These homozygotes are usefu l in genetic studies and for further improvement of the crop species . This utility of haploids for producing homozygous breeding lines from heterozygous parents is well documented .

However, although used successfully for producing haploid plants in many species, the technique has not been successful for species of Corchorus. Anther callus induction and root formation from callus have been reported in Corchorus 1

'2 but plant regeneration

from anther derived calli of some cu lti vars, wild and hybrid derivatives of Corchorus spec ies, failed even after repeated subculturing in different mediav. Thi s failure may be because of some limiting factors which affects pollen embryogenesis in vitro, but the effects of various factors on the induction of pollen

'Part of Ph. D thesis , Department of Agricultural Botany, University of Read ing, England. "Present add ress: Breeding Divi sion. Bangladesh Jute Research Institute, Manik Mia Avenue, Dhaka 1207 , Bangladesh.

embryogenesis or organogenesis in Corchorus were not reported. A protocol for anther and rnicrospore culture has not been established, and the present study of the influence of some factors on mtcrospore embryogenesis in Corchorus was an attempt to develop a technique for haploid production in Corchorus.

Materials and Methods Plant materials- Corchorus olitorius Taiwan was

used with a genotype obtained from Taiwan. Seeds were sown separately at different times in 23 em plastic pots filled with a mixture of loam, peat and sand (7 :6:6). The pots were kept in the glasshouse at an average temperature of 28°C. The glasshouse provided a 16 hours photoperiod with day and night temperatures of 32° and 25°C respectively. The regular watering was supplemented with a nutrient feed solution of nitrogen, potassium & phosphorus(2: I: I ) twice in a week.

Selection of bud size-Buds of different sizes collected from the mai n inflorescence were squashed on a slide, and after removal of debri s vverc stained with safranine and examined with a light microscope. For each bud size 5 anthers were squashed separately, and on the basis of observation of microspores buds were classified into early uninucleate, mid uninucleate, late uninucleate and binucleate stages. Buds 2-3mm in length with anthers in mid uninucleate stage of pollen development containing

594 INDIAN 1 EXP BIOL, JUNE 2000

mostly uninucleate m1crospores were used for microspore isolation.

Microspore isolation-For each experiment 20 anthers from each of I 0 buds were squashed in a Petri dish (60 x 15 mm) in two drops of liquid MS medium4

without growth hormones under sterile conditions5.

To the mixture of microspore and anther wall debris, 5 ml of liquid MS medium was added and the solution was stined very carefully with a sterile glass rod for 2-3 min to isolate microspores which remained strongly attached with the mucilage that occurred within in anthers. Using the technique described by Lichter6 microspores were isolated and microspore suspensions of 1.25 ml of medium with an average number of 2 x I 04 microspores were cultured in each Petri dish (60 x 15 mm) .

Microspore culture - Various medi a have been used successfully for microspore and anther culture7

·8

.

Isolated microspores of C. olitorius Taiwan were cultured in four media, NN,9 NLN,6

'10· B5

11 and A-751 2. Several other media were also used ; MS medium supplemented with 2 mg/L cytokinin, either zeatin or BA, and either 0.1 or 0 .5 mg/L IAA; 25.0 mg/L colchicine and I % activated charcoal. Growth hormones and colchicine were filter sterilized and added to the media after autoclaving. There were eight Petri dishes for each of the culture media. All cultures were incubated in the dark at an incubation temperature of 35°C for one day and then transferred to a cul ture room in dark at an average temperature of 25° ± 2°C. Data were analyzed for the analysis of variance (ANOV A).

Anther culture - Media used for anther culture were basic MS supplemented with different concentrations of sucrose (3, 5, I 0 & 13%) and with 2.0 mg/L zeatin and 0.1 mg/L IAA. Three temperature treatments, 25°C continuously, 30°C for 20 days and then at 25°C continuou sly, and 35°C for I day and then at 25°C continuou sly, were also used with MS media supplemented with 2.0 mg/L zeatin and 0.1 mg/L IAA. All cultures were incubated in the dark with five replications of each treatment. Data were analyzed for the analysis of variance (ANOV A).

Results and Discussion

Microspore culture Embryogenic induction- Microspores from the

mid uninucleate stage of pollen development produced a few multicellular proembryoids. Within 48 hr of culture incubation some microspores divided symmetrically producing two identical cells in

contrast to the asymmetrical divis ion in normal development. This first divis ion is very important in switching the development in isolated microspores to embryogenesis. Very few microspores developed further after one or two di visions. In some cases, the divis ion of microspores continued and multicellular proembryoids were formed. These proembryoids did not deve lop after the 8 - 12 celled stage but produced microcalli. The cells of some of these microcalli divided repeatedly and formed an elongated mass of divid ing cells, but after three to four weeks this divi sion and development stopped . Later, in a few cases, the multicellular microcalli were observed to undergo repeated divi sion and finally produced a large mass of microspore callus .

Effect of media-Four microspore culture media, NN9

, NLN6•10

, B511

, A-75 12 (except MS) reported to be suitable for other crop species were tested for Corchorus microspore culture. Response of microspore division occurred only in NLN medium and was observed in only 0. I % of the microspores. However, further divi s ion and deve lopment of these microspores did not occur in thi s medium. As the response of microspores in NLN medium was very low and insignificant, further attempts were made with basic MS media with various combinations of cytokinins and auxins, and tempe rature, sucrose, colchicine and charcoal treatments .

Effect of growth regulators- The results on the effect of various growth regulators (Table I) showed that the basic MS medium supplemented with IAA and either zeatin or BA played an important role on induction of microspore division. In all of the media microspores showed some division, but in low frequencies. Initially, about I 0 -30% microspores had one or two divi sions. The highest number and the highest frequency of dividing microspores were on medium with 2.0 mg/L zeatin and 0 .1 mg/L IAA. About 0.2 - I % of the dividing microspores continued di vis ion and development and produced e ither a many celled proembryo or microcalli . The numbers of multicellular microcalli were higher in this medium with zeatin and IAA than in medium with BA and IAA. After repeated division a few of these microcalli produced a many celled microspore callus, and thi s occuned only on medium with 2.0 mg/L zeatin and 0 .1 mg/L IAA.

Other treatments- The addition of I% activated charcoal with basic MS medium supplemented with either zeatin or BA and 0.1 mg/L IAA did not enhance embryogenesis. Colchicine at a concentration

ALI & JONES: MICROSPORE CULTURE IN CORCHORUS 595

of 25 mg/L was shown to be stimulative for microspore embryogenesis in Brassica napus13

·

increasing both the number of microspores induced to divide and the number of mjcrospores producing embryos. Colchicine in the same concentration when added with basic MS medium supplemented with either 2.0 mg/L zeatin and 0.1 mg/L IAA or 2.0 mg!L BA and 0.5 mg/L IAA appeared to reduce the frequency of dividing microspores in Corchorus.

Temperature and sucrose treatments on microspore callus formation from anthers.

Validity of this experimental approach Several factors which are known to effect pollen

embryogenesis or organogenesis, e.g. pollen development stage, culture media, sucrose treatment, cold treatment, temperature treatment, effect of L­glutamine and glutathione, colchicine treatment and ethanol and water treatment, have been studied with anthers of Cm·chorus. None of these treatments were successful for pollen embryo formation from anthers. However, two treatments, temperature and sucrose, showed some significant effects on microspore callus formation from cultured anthers. As development of microspores free in culture parallels very closely their development in cultured anthers 1\ these two treatments have been included to report their positive effects on the embryogenic development of m1crospores.

Effect of temperature- Temperature treatments have been used successfully to improve anther culture

efficiency in Brassica14 and Triticum15 and three temperature treatments were used in anther culture of Corchorus (Table 2). It was observed that the incubation temperatures showed some significant effects on microspore callus formation. Two temperature treatments, both continuously in the dark, 30°C for 20 days and then at 25° and 35° C for I day and then at 25°C increased markedly the frequency of anthers producing microspore callus . The former treatment showed a problem of media hardening during the period of 20 days, but in the latter there was no such problem. It would be worthwhile to use this latter treatment and possibly other similar treatments for the induction of first microspore division in C. olitorius.

Effect of sucrose-Microspore callus formation occurred on all the media with sucrose, but in different frequencies. Comparatively, the highest numbers and the highest frequencies of anthers producing calli were on media with 3% and 5% sucrose (Table 3) . MS media with I 0% and 13% sucrose showed a much lower anther response. The effects of media on the number of anthers producing microspore calli and the frequency of anthers producing these calli were highly significant (?>0.001) .

Callus subculture-Subculturing of microspore calli obtained in different experiments, either in a fresh preparation of the same media, or in different regeneration media containing various concentrations of a cytokinin, either zeatin or BA or kinetin, without or with auxin, and also on medium without growth hormones, did not enhance regeneration of shoots .

Table I -Micros pore response in Corchorus olitorius Taiwan on basic MS medium supplemented with zeati n (Z), BA (BA) and IAA (I) with or without colchicine

(C).Media (mg/L)

Media (mg/L) Microspore response Z-1 Z-IC BA-1 BA-IC

No. of microspores counted 2080 2250 2200 2400 No. of dividing microspores 586 240 436 3 15 Frequency of dividing micros pores 0.3 0.1 0.2 0.1 No. of microcalli 7 3 I 0 Frequency of microcalli 00.1 0.01 0.002 0 No. of calli 2 0 0 0 Frequency of callus formation 0.3 0 0 0

Table 2- Callus induction from cu ltured anthers after temperature treatments (MS medium supplemented with 2.0 mg/L zeatin and 0.1 mg/L IAA).

No. of anthers cultured No. of anthers producing calli Frequency of anthers producing calli

Temperatures ("C) 25" continuous 30° for 20 days 35" for I day

80 8

0.1

then 25 then 25"

95 40 0.4

85 31 0.4

596 INDIAN 1 EX P BIOL, JUNE 2000

Table 3 - Microspore callus formation from anthers at four sucrose levels (MS media supplemented with 2.0 mg/L zeatin and 0.1 mg/L IAA).

Characters

No. of anthers cultured No. of anthers producing calli Frequency of anthers producing call i

3 102 26

0 .3

Sucrose levels(%) 5 10

82 112 29 6

0.4 0 .05

13 120

12 0.09

Table 4- Root fo rmat ion from microspore callus in Corchorus : number (No.) and frequency (Freq) of call i producing roots on MS medi a supplemented with various levels of cytokinin (mg/L) without or with 0. 1 mg/L IAA)

Character lA A Zeatin

0 2 4

No. 0 3 2 I 0 Freq . 0.4 0 .2 0.1 0 No. 0. 1 4 3 2 0 Freg . 0 .5 0.4 0 .2 0

Rooting from callus - Further callus growth was observed on most of the subculture media, and some calli produced many roots (Table 4). There were di fferences in root formation in different medi a, and the highest number of calli produc ing roots was on medium with 0.1 mg/L IAA without cytokinin . Root formation from callus also occurred on other media with either 1.0 or 2.0 mg/L zeatin or 1.0 mg/L BA or 1.0 mg/L kinetin , each without or with IAA. Root formation did not occur on media with either 4.0 mg/L of any one of the cytokinins or 2.0 mg/L BA. Further subculture of calli on fresh preparations of media with high concentrations (4.0 mg/L) of zeatin, BA or kinetin , did not enhance regeneration .

Discussion The results of the present study are in agreement

with the previous reports 1•3 in respect of anther callus

formation, but the response of isolated microspores in vitro culture has not been reported previously. This paper report the effects of the various factors in Corchorus olitorius microspore culture. The study confirms that isolated microspores of the cultivated species of Corchorus olitorius when culti vated on bas ic MS medium supplemented with zeatin and IAA show some division and development and produce microspore callus. This demonstrates a "switch" from gametophytic to sporophytic pathway of pollen development in vitro in Corchorus. Development of callus was dependent on the medium and on temperature. The combination of 2.0 mg/L zeatin and 0.1 mg/L IAA with either 3% or 5% sucrose in a basic MS medium at incubation temperatures of 35°C for I day and then 25°C continuously, was most effective for induction of microspore division and callus formation in these experiments with this genotype of C. olitorius.

C tokinins BA Kinetin 2 4 2 4

I 0 0 I 0 0 0.1 0 0 0.1 0 0 2 0 0 2 0 0.2 0 0 0 .2 0 .1 0

Root formation from m1crospore callus demonstrates the development of meristems in the callus, but the absence of any shoot formation confirms that thi s genotype of Corchorus, and possibly all genotypes of the spec tes, have recalcitrance to organogenesis. Further improvement of the technique by manipulation of growth hormones, carbohydrate and temperature, and by using a number of different genotypes, may enable the deve lopment of an effective method for producing haploid plants of Corchorus . These limited results may be useful in the planning of any further attempts.

Acknowledgement The Financial support of a Technical Training

Award Administrated by The Bri tish Council is acknowledged.

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