Effect of spikelet position on rice anther culture efficiency
Rownak Afza, Mei Shen, Francisco Javier Zapata-Arias *, Jiahua Xie,
Haji Khamis Fundi, Kang-Seop Lee, Eva Bobadilla-Mucino, Andrea Kodym
Plant Breeding Unit,FAO/IAEA Agriculture and Biotechnology Laboratory,International Atomic Energy Agency Laboratories,
A-2444Seibersdorf,Austria
Received 6 September 1999; received in revised form 1 December 1999; accepted 3 December 1999
Abstract
The potential of anthers from different parts of the panicle to induce callus was investigated with thejaponicarice variety Taipei 309. The results showed that the callusing abilities of anthers from different spikelet positions were significantly different. After plating 4483, 4496, 4348 anthers from the basal, middle and top parts, the percentage of anthers forming calli was 20% in the basal part, 12% in the middle part and 8% in the top part. The anthers of basal parts containing pollen at all uninucleate stages, including early, middle and late, showed higher callus induction frequency than those from middle and top parts. The green plantlet regeneration frequencies of top, middle and basal spikelets were around 18% in all three cases. From the results it would appear that anthers from the basal part of the panicle should be used in anther culture of rice in order to obtain higher efficiencies, and thereby optimise the usefulness of this technique in rice breeding programmes. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords:Anther culture; Morphological marker;Oryza sati6aL.; Pollen stage
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1. Introduction
In plant breeding, the production of haploid plants speeds up the breeding cycle by fixing ho-mozygosity in one generation. It allows an in-crease in selection efficiency due to better discrimination between genotypes within any gen-eration and efficient retention of desirable genes in later generations [1]. The acceleration of the breed-ing cycle and increase of selection efficiency make double haploid techniques very attractive, not only for conventional breeding but also for plant im-provement through mutation induction. Anther culture technique can be considered complemen-tary to mutation induction because both dominant and recessive genes will be phenotypically ex-pressed allowing easier isolation of desirable reces-sive mutations.
While the anther culture technique is widely used for practical breeding, its application is still limited by many factors which influence culture efficiency, such as the genotype of the explant [2,3], the growing conditions of the donor plants [4], the developmental stage of the microspores [5,6], pre-treatment of panicles [7], the culture methods [4,8], the media [4,9] and the culture conditions [7,10]. One of the major constraints in the use of anther culture in rice improvement programmes is the identification of responsive an-thers for callus formation. Little work has been reported on the use of morphological markers such as panicle length, spikelet position, spikelet colour, anther colour and anther position in the spikelet on subsequent callus induction. Based on the study by Mercy and Zapata [11], the distance between the flag leaf and the subtending leaf as well as the late uninucleate and early binucleate pollen stage has been used as markers for callus induction although with inconsistent success. Abbre6iations: BA, 6-benzylaminopurine; 2,4-D,
2,4-dichlorophe-noxyacetic acid; NAA,a-naphthalene acetic acid.
* Corresponding author. Fax: +43-1-260028222.
E-mail address:f.zapata-arias@iaea.org (F.J. Zapata-Arias)
Shahjahan et al. [12] observed that the best mi-crospore stage for highest callus induction in four indica rice varieties was the mid-uninucleate stage and this corresponded with spikelets of yellowish green colour and anthers reaching the middle of the spikelet. Yin et al. [13] using a japonica culti-var observed that the best microspore stage for callusing was the late-uninucleate stage when spikelets were yellowish green in colour and the length of stamen was 1/3 to 1/2 of the glume.
The present study was focused on the effect of the spikelet position within the panicle on callus induction and plant regeneration. This was done in order to provide breeders with practical guide-lines to make rice anther culture more convenient and efficient. The distance between flag leaf and subtending leaf was used as a marker for harvest-ing the panicle. The developmental stages of pol-len derived from anthers from different positions of the spikelet in the panicle were also identified in order to correlate this with spikelet position and callus formation.
2. Materials and methods
2.1. Plant material
The japonica rice variety Taipei 309 (Oryza sa -ti6a. L), which had been proven to be well
re-sponsive to anther culture [14], was used in the present study. Plants were grown in pots in the greenhouse during the months of April to August with an average day/night temperature of 30/
20°C and a minimum relative humidity of 60%. Panicles were harvested at the booting stage with the distance between the subtending leaf and the flag leaf being 7 – 10 or 11 – 13 cm. The panicles were subjected to a cold pre-treatment at 8°C for 8 days and then were surface sterilised by im-mersing in 70% ethanol for 20 s and then 10% Clorox (commercial bleach containing 5.2% (w/v) NaOCl) for 10 min, and washed three times with sterile distilled water before plating. Each panicle was cut into three equally long parts: top, middle and basal. Meanwhile, one spikelet of each part was randomly selected from each panicle and fixed in acetic-ethanol (1:3) as mordant for 24 h, then stained with 2% aceto-carmine and exam-ined under a light microscope to identify the pol-len stages present.
2.2. Callus induction
The anthers from each part were plated asepti-cally at a density of about 40 anthers per 50 mm petri dish (Bibby Sterilin, UK) containing semi-solid N6 medium [15] supplemented with 2 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D) and
60 g l−1 sucrose (pH 5.8). The medium was
soli-dified with 5 g l−1 agarose. The dishes were
la-belled with the panicle number as well as the portion (top, middle, base), sealed with parafilm and incubated in the dark at 2592°C for callus induction. A total of 116 panicles was used. In the 7th week after inoculation, callus induction frequency was calculated on the basis of the number of anthers producing callus. Some an-thers produced more than one callus, but for this calculation, all calli originating from one anther were considered as one.
2.3. Plant regeneration
Calli with a size of at least 2 mm were trans-ferred to Murashige and Skoog [16] basal semi-solid medium, supplemented with 1 mg l−1
6-benzylaminopurine (BA), 0.5 mg l−1
a
-naph-thalene acetic acid (NAA) and 30 g l−1 sucrose
(pH 5.8), and were incubated at 2592°C under a 12 h light photoperiod supplied by cool white fluorescent lamps (66 mmol m−2 s−1) for plant
regeneration. Similar numbers of calli derived from the three spikelet positions were plated, 634 derived from the top, 705 from the middle and 655 derived from the basal position. Regenerated plants were counted on the basis of the number of callus producing plantlets. If more than one plant originated from the same callus, they were considered as only one.
3. Results
3.1. Callus induction
cm. The callusing abilities of anthers from differ-ent panicle positions were significantly differdiffer-ent (P=0.01), with anthers from the top, middle and basal part showing 7.52, 12.43 and 20.01% callus-ing efficiencies, respectively.
It has been reported that the pollen stage of plated anthers can seriously affect the callus for-mation [11,17,18]. Within one panicle, pollen in anthers from the top part are the oldest, and in the basal part the youngest. In order to correlate pollen stage with spikelet position and callus for-mation, the pollen stage of the three parts of each panicle were determined. Table 1 shows the effects of pollen stage of plated anthers from the three parts of the panicle on callus induction. Among the sampled anthers, 87.74% were at the uninucle-ate stage, 10.35% anthers at the binucleuninucle-ate stage, and only 1.9% anthers were at the tetrad stage. In the present study, 95.5% of the responding anthers were at the uninucleate stage (1703 an-thers). Anthers at the tetrad stage were too young to produce callus. At the early, middle and late uninucleate stage anthers showed different re-sponse. The rate of responding anthers at the late uninucleate stage was the highest with 16.12%, followed by the early uninucleate stage with
15.35% and the middle uninucleate stage with 11.71%. However, the anthers with pollen in the same developmental stage but derived from differ-ent panicle positions exhibited differdiffer-ent callusing ability. The results indicated that anthers from the basal parts with pollen at any uninucleate stage had a higher callus induction frequency than from the middle and the top parts. The callus formation frequencies of anthers from the top, middle, basal parts at the early uninucleate stage were 6.22, 11.50, 19.97%, respectively, from the middle unin-ucleate stage 7.67, 12.07, 16.23% and from the late uninucleate stage 9.91, 14.85, 28.93%, respectively. The highest percentage of callusing anthers was from the basal parts with pollen in the late uninu-cleate stage (28.93%). However, this correlation between spikelet position and callus production was not observed when binucleate anthers were used, in which case frequency of callus production was low for anthers from both top and basal positions.
3.2. Plantlet regeneration
After about 3 weeks on regeneration medium, green points appeared on the callus. The frequency
Table 1
Effect of pollen stage of plated anthers from the three parts of the panicle on callus response (%)
All stages Early uninucleate Mid-uninucleate Late uninucleate Binucleate
Plated % Re- Plated %
Re-Response
Plated Plated % Re- Plated %
Re-sponse sponse
sponse sponse
No. %
327c* 7.52 498 6.22 1265 7.67 1686 9.91 819
Top 4348 3.91
4496 14.85 407 10.80
Middle 559b 12.43 1504 11.50 1077 12.07 1428
Basal 4483 897a 20.01 2233 19.97 1041 16.23 961 28.93 153 2.60
13 327
Total 1783 13.38 4235 15.35 3383 11.71 4075 16.12 1379 5.80
* The letters a,b and c refer to significant differences at the 0.01 level of an LSD Test using M-Stat.
Table 2
Plantlet regeneration of anther-derived calli from different spikelet positions
No. callus plated
Spikelet position No. callus regenerating % of callus regenerating
Green plantlets In total
Top 634 163 25.71 17.82
705 152 21.56 18.01
Middle
655 136 20.76 17.71
of plant regeneration for calli from different pani-cle positions is presented in Table 2. The average regeneration frequency of plated calli was 22.61%, including green and albino plants. No significant difference in green plant regeneration frequency was observed among the calli derived from differ-ent parts; frequencies of top, middle and basal were 17.82, 18.01 and 17.71%, respectively.
4. Discussion
According to Mohapatra and Sahu [19], the distribution of soluble carbohydrates, free amino acids and soluble phosphates were not uniform within rice panicles at the time of anthesis and early stage of panicle development. The concentra-tion of soluble carbohydrates, free amino acids and soluble phosphates increased from the base to the top and then declined with time until maturity. Sahu and Mohaptra [20] studied the spikelet devel-opment in rice panicles in relation to assimilates of primary branches. They demonstrated that the presence of a higher concentration of assimilates did not help the spikelet survive on the proximal branches anymore than that on the distal. They also found that 2 weeks prior to anthesis, the gradient in the concentration of assimilates of the primary branches increased acropetally, i.e. from bottom to the top. During the following week, however, the situation reversed and a higher con-centration was found in the lower branches than the upper part. They reported that the concentra-tion of amino acids and phosphates changed sig-nificantly with time during the period prior to anthesis and maturity. According to Xu and Ver-gara [21], the development of the primary branches was basipetal, i.e. growth from apex to the base but spikelet development on a branch was acropetal.
The present study reported that the anthers in different spikelet positions within one panicle had significantly different callusing abilities. Anthers from the top part had the lowest callus formation frequency while those from the basal had the highest, even when anthers contained pollen at the same development stage. According to the above discussion there is a higher concentration of assim-ilates, amino acids and carbohydrates in the basal part than in the top part during the development of the panicle. This unequal quantitative
distribu-tion of the amino acids and other assimilates within a panicle might correlate with callusing ability of anthers derived from spikelets of differ-ent positions in the panicle. It has been already reported by Bei [22] that the distribution of en-dogenous hormones and amino acids of donor plants might influence the callusing ability of an-thers derived from different parts. Xie et al. [23] reported that the quantity of amino acids is im-portant for switching from gametophytic to sporo-phytic development followed by callus formation. In this study, anthers derived from different positions, but with pollen in the same development stage possessed different callusing ability. Accord-ing to Arai and Kono [24], meiosis of pollen was found to occur in anthers of the same length but that meiosis of the upper spikelet occurred when the lemma length was smaller than the lower spikelet regardless of spikelet position in the pani-cle. They assumed that the positional difference in the spikelet growth is not only dependent upon the distribution of translocated substances but also depended upon the physiological specificity of each organ of the spikelet.
From the present investigation it can be con-cluded that by culturing anthers from the basal part of the panicle a higher callusing response can be achieved. The unequal distribution of assimi-lates within the panicle and the different physio-logical developmental stage of the spikelets could be the reasons for the differences in callusing ability of anthers from spikelets of different posi-tions in the panicle, in addition to pollen stage.
Acknowledgements
Many thanks to A. Draganitsch, F. Zwiletitsch and G. Berthold for their technical assistance and to Mr Rigney and the staff of the Plant Breeding and Genetics Sub-programme for their critical reading of the manuscript.
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