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Embryogenesis and plant regeneration from anther culture in loquat (Eriobotrya japonica L.)
Available online at www.sciencedirect.com
Scientia Horticulturae 115 (2008) 329–336 www.elsevier.com/locate/scihorti
Embryogenesis and plant regeneration from anther culture in loquat (Eriobotrya japonica L.) Li Junqianga,b, Wang Yongqinga,*, Lin Lihuab, Zhou Lijunb, Luo Nana, Deng Qunxiana, Xian Junrena, Hou Chunxiaa, Qiu Yuana a
College of Forestry and Horticulture, Sichuan Agricultural University, Ya’an 625014, Sichuan, China Department of Bioengineering, Yibin Vocational & Technical College, Yibin 644003, Sichuan, China
b
Received 24 October 2006; received in revised form 30 September 2007; accepted 4 October 2007
Abstract The object of this study was to induce embryogenesis and establish plant regeneration system for anther culture in loquat (Eriobotrya japonica L.). Cold pretreatment was a key factor, and supplement of 2,4-D in the media was absolutely necessary for induction of calluses from cultured loquat anthers. The best response of anthers to in vitro culture was obtained when a 48-h cold pretreatment was employed to flower buds at 4 8C in darkness. Genotype was a decisive factor for embryo differentiation. When anther-derived calluses of three loquat cultivars, i.e., cv. ‘Longquan1’, ‘Dawuxing’ and ‘Zaozhong6’, were transferred to embryo differentiation medium, embryos were induced only for cv. ‘Dawuxing’ on MS medium containing 3% sucrose, 0.23 mM ZT in combination with 0.05 mM NAA + 0.05 mM IBA or 0.11 mM NAA + 0.10 mM IBA, and the differentiation rates were 3.33% and 10.00%, respectively. The results of histological studies showed that embryos developed through typical globular, heart, torpedo and cotyledon stages after 4 weeks of culture. The treatment designed to mature the embryos on medium containing 3% of sucrose at 4 8C under darkness for 4 weeks was effective for subsequent embryo germination and plant conversion, which gave rise to 72.5% plant recovery. Cytological studies showed that 26 plantlets were haploids (n = 17) and the remaining 4 plantlets were diploids for the 30 regenerants tested. # 2007 Elsevier B.V. All rights reserved. Keywords: Anther culture; Callus induction; Embryogenesis; Eriobotrya japonica L.; Haploid; Plant regeneration
1. Introduction Plant breeders have always been interested in shortening the period required for breeding. But in traditional breeding systems, more than six generations are needed for homogenizing alleles for a particular trait (Rimberia et al., 2005). At haploid level, every gene is hemizygous. After chromosome doubling, which makes an identical copy of each haploid chromosome, every genotype becomes homozygous (Baenziger, 1996). Thus, production of haploids offers new possibilities for genetic studies and for an increased efficiency in selection. Anther culture, originally developed by Guha and Maheshwari (1964, 1966), allows rapid production of haploids, and
Abbreviations: BA, benzyladenine; 2,4-D, 2,4-dichlorophenoxyacetic acid; IBA, indolebutyric acid; KT, kinetin; MS medium, Murashige and Skoog medium (1962); NAA, a-naphthaleneacetic acid; PGRs, plant growth regulators; TDZ, thidiazuron; ZT, zeatin. * Corresponding author. Tel.: +86 835 2882782; fax: +86 835 2882578. E-mail address: [email protected] (Y. Wang). 0304-4238/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2007.10.007
subsequently chromosome doubling can lead to completely homozygous diploid plants. This technique has become one of the major sources of haploid plant production used to develop homozygous diploids in plant breeding programs (Bajaj, 1983). Compared with conventional inbreeding, the in vitro androgenesis technique enables a faster generation of virtually fully homozygous lines (Aulinger et al., 2003). Androgenesis is a valuable procedure for obtaining haploids and doubled haploids in selection and breeding programs, especially for woody species that are heterozygous (Radojevic et al., 2000). Nevertheless, there are a number of factors that affect androgenesis, including genotype and physiological state of the donor plant, anther age and pollen developmental stage, pre-culture treatment, physical factor (temperature, light, atmospheric condition, anther density and orientation and pH) and chemical factor (culture medium, sugar and plant growth regulator) (Sopory and Munshi, 1996). As a result, so far, there have been only a limited number of successes of anther culture in woody species. As an important economic fruit crop, loquat (Eriobotrya japonica L.) is widely cultivated between 208 and 358 latitude
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including China, Japan, India, Pakistan, Madagascar, Reunion Island, Mauritius Island, the Mediterranean countries (Spain, Turkey, Italy, Greece, Israel), the United States (mainly California and Florida), Brazil, Venezuela, and Australia (Badenes et al., 2000; Vilanova et al., 2001). This crop is characterized by a long reproductive cycle and juvenile phase (both of several years). Because of heterozygosity and long juvenile period of the plant, only a limited number of genetic studies have been performed and no long-term breeding program has ever been established for this crop. The traditional breeding methods are time-consuming and limited by space required for field experiments. Consequently, there is a need to shorten the breeding period using techniques such as haploid induction via anther culture. The present paper reports, for the first time, the establishment of plant regeneration system and haploid production through embryogenesis from anther culture of loquat. 2. Materials and methods
combination with a-naphthaleneacetic acid (NAA) + indolebutyric acid (IBA) (0.05 + 0.05, 0.11 + 0.10 and 0.21 + 0.20 mM). The conical flasks were capped with non-absorbent cotton plugs wrapped with one layer of cheesecloth. The media were adjusted to pH 5.6 prior to addition of agar and sterilized at 122 8C and 104 kPa pressure for 17 min. For callus induction, cultures were maintained at 25 1 8C in the dark. For embryo differentiation, cultures were cultured in a culture chamber at 25 1 8C under 16 h daily illumination with 2000 lx fluorescent light. 2.2. Histological study Embryos in different developmental stages were fixed in FAA (70% ethanol:formalin:acetic acid, 18:1:1, v/v/v), rinsed, dehydrated and embedded in paraffin (56–58 8C) using classical techniques. Sections (10 mm thick) were cut using a rotary microtome, and stained with haematoxylin. Then, sections were observed under a light microscope and documented by a digital camera (Motic BA200).
2.1. In vitro culture of anthers 2.3. Embryo maturation, germination and plant conversion Anthers of loquat (E. japonica L.) cv. ‘Longquan1’, ‘Dawuxing’ and ‘Zaozhong6’ were the experimental materials in the present investigations. The mother plants were grown in the experimental plots using standard agronomic practices. Flower buds (5–6 mm in diameter) were harvested, which contained approximately 80% uninucleate microspores confirmed by periodically microscopically checking the microspore stage. The flower buds were collected in a conical flask with a piece of wet cotton to maintain a high humidity and kept at 4 8C in dark for 0 (control), 24, 48, 96 and 144 h. Before culturing of anthers, the thick floss of the sepals was completely scraped off (otherwise they would make it difficult to sterilize the flower buds) with a surgical scalpel and then washed in distilled water containing 3% (v/v) Tween (detergent) for 5 min. The final step of sterilization and further operations were carried out in a laminar air-flow cabinet under aseptic conditions. The flower buds were surface-sterilized by immersion in 0.1% (w/v) mercuric chloride solution with periodic agitation for 8 min, and washed with sterile distilled water for five times. After removing filament, the intact anthers were plated horizontally, with the connective tissues contacting the medium, in the conical flasks (150 ml) containing 30 ml of medium. Callus induction medium consisted of Murashige and Skoog (MS) (1962) mineral salts and vitamins, 7% (w/v) sucrose, 0.6% (w/v) agar, and it was supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) (0.05, 0.09, 0.23, 0.45, 1.36, 2.26 and 4.52 mM) and benzyladenine (BA) (8.90 mM). Calluses which were derived from MS medium containing 7% (w/v) sucrose, 0.6% (w/v) agar, 2.26 mM 2,4-D and 8.90 mM BA (a 2-day cold pretreatment was employed to these cultured anthers) were used to achieve embryo differentiation. The successfully induced calluses were separated from anther walls and cultured on an embryo differentiation medium. Embryo differentiation medium consisted of MS mineral salts and vitamins, 3% (w/v) sucrose, 0.6% (w/v) agar, and zeatin (ZT) (0.05, 0.23 and 0.46 mM) in
The effect of various maturation treatments was evaluated in terms of subsequent embryo germination and plantlet conversion ability. White opaque cotyledonary embryos were isolated from embryo clusters and cultured on different maturation media, consisting of PGR-free basal medium supplemented with sucrose (3% or 5%), and stored at 4 or 25 8C for 4 weeks under darkness. After 4 weeks of culture on maturation medium, embryos were transferred to MS basal medium with 3% sucrose. And then, they were transferred into Petri dishes containing 20 ml germination medium (basal MS medium) for 4 weeks. Photos of callus induction, embryo differentiation, embryo maturation, embryo germination, plantlet conversion were taken using a stereoscopic microscope (Motic DM143) and a digital camera (SONY F717). 2.4. Transplantation of plantlets and cytological studies Well-developed plantlets were removed from culture vessels and washed in distilled water to remove traces of the medium. These plantlets were then transplanted to plastic cups containing a mixture of autoclaved peat, perlite and garden soil (1:1:1). The plantlets were grown in a growth chamber (25 1 8C and 16 h photoperiod of 2000 lx illumination, 90% relative humidity). After 8 weeks, these plants were transferred to the greenhouse for further growth. For cytological studies, the root tips of 30 randomly selected regenerants were harvested and treated with 2 mM aqueous solution of 8-hydroxyquinoline at room temperature for 4 h. The pretreated root tips were fixed in acetic alcohol solution (3 parts of ethanol:1 part of glacial acetic acid) for 24 h. Subsequently root tips were washed in distilled water and hydrolyzed in 1N HCl at 60 8C for 10 min and stained with Feulgen reagent for 1 h and squashed in 45% acetic acid (Armstrong, 1996). Chromosome preparations were photographed using a light microscope (Motic BA200).
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twice. Cultures were observed periodically and morphological changes were recorded at regular intervals of 4 weeks. The term of maturation in this study denotes the development of globular and heart-shaped embryos into distinct cotyledonary form which is a bipolar embryo with well-developed cotyledons, and the term germination means the development of shoots or roots or both shoots and roots (plant conversion). The results presented in this study are the mean of 300 anthers, 30 anther-derived calluses or 20 embryos per experiment. The data were subject to analysis of variance (ANOVA) to assess treatment differences and interactions using the SPSS statistical package for windows (release 11.0, SPSS INC). Significance between means was tested by Duncan’s multiple range test.
2.5. Data collection and analysis A factorial design was employed for callus induction experiments and a randomized complete block design was used for the other experiments. For callus induction, 300 anthers were cultured (20 anthers per conical flask and 15 replicates per treatment). For embryo differentiation, 30 calluses were cultured (10 calluses per conical flask and 3 replicates per treatment). For embryo maturation and germination experiments, each treatment was applied to 20 embryos (10 embryos per conical flask and 2 replicates per treatment). Callus induction and embryo differentiation experiments were repeated three times, and embryo maturation and germination experiments were repeated
Table 1 Effect of cold pretreatment to flower buds and PGRs on the callus induction of anthers after 4 weeks of culture in three loquat cultivars Cold pretreatment (h)
2,4-D (mM)
Anthers forming callusesa cv. ‘Longquan1’
cv. ‘Dawuxing’
cv. ‘Zaozhong6’
No.
No.
No.
%
0
0.05 0.09 0.23 0.45 1.36 2.26 4.52
0 11 0 0 2 0 5
0 3.67 0 0 0.67 0 1.67
24
0.05 0.09 0.23 0.45 1.36 2.26 4.52
15 31 55 68 77 88 85
5.00 10.33 18.33 22.67 25.67 29.33 28.33
48
0.05 0.09 0.23 0.45 1.36 2.26 4.52
20 34 55 58 37 208 168
96
0.05 0.09 0.23 0.45 1.36 2.26 4.52
114
0.05 0.09 0.23 0.45 1.36 2.26 4.52
Significance of two-way ANOVA 2,4-D concentration (A) Cold pretreatment (B) AB a **
%
5 0 3 0 3 0 0
1.67 0 1.00 0 1.00 0 0
F E D C B A A
18 33 34 37 47 46 45
6.00 11.00 11.33 12.33 15.67 15.33 15.00
6.67 11.33 18.33 19.33 12.33 69.33 56.00
E D C C D A B
23 39 55 55 52 169 151
3 7 15 20 32 53 45
1.00 2.33 5.00 6.67 10.67 17.67 15.00
E DE CD C B A A
1 5 6 12 16 27 22
0.33 1.67 2.00 4.00 5.33 9.00 7.33
C C C B B A A
%
3 0 6 0 2 0 1
1.00 0 2.00 0 0.67 0 0.33
C B B B A A A
38 37 52 53 61 72 69
12.67 12.33 17.33 17.67 20.33 24.00 23.00
D D C C BC A AB
7.67 13.00 18.33 18.33 17.33 56.33 50.33
E D C C C A B
44 59 73 103 79 256 226
14.67 19.67 24.33 34.33 26.33 85.33 75.33
F E D C D A B
2 2 7 22 53 61 53
0.67 0.67 2.33 7.33 17.67 20.33 17.67
D D D C B A B
9 18 25 41 59 132 127
3.00 6.00 8.33 13.67 19.67 44.00 42.33
E DE D C B A A
2 7 8 9 17 23 22
0.67 2.33 2.67 3.00 5.67 7.67 7.33
20 39 39 83 81 95 92
6.67 13.00 13.00 27.67 27.00 31.67 30.67
D C C B B A A
D CD C C B A AB
**
**
**
**
**
**
**
**
**
Means having the same letter in the columns were not significantly different according to Duncan’s multiple range test (P = 0.01). Significant at P = 0.01.
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3. Results 3.1. Effect of cold pretreatment of flower buds and PGRs on callus formation of cultured anthers The results of callus induction experiments were summarized in Table 1. Analysis of variance of the callus induction rate indicated that callus formation was significantly affected by cold pretreatment (P = 0.01), 2,4-D concentration (P = 0.01), and their interaction (P = 0.01). If the anthers were not coldtreated, callus induction rates were not significantly affected by 2,4-D concentration (P = 0.05). A 48-h cold shock was beneficial as it significantly increased the induction rate of
calluses, reached the apex of 69.33%, 56.33% and 85.33% for cv. ‘Longquan1’, ‘Dawuxing’ and ‘Zaozhong6’, respectively. Prolonged cold shock (96–144 h) dramatically decreased the potential of anthers to produce calluses because it increased the frequency of anthers to turn brown and then became necrotic. Regardless of the time of cold pretreatment used to the flower buds, the best results in terms of callus induction were obtained with the higher concentration of 2,4-D (2.26 mM). The responding anthers grew larger in size and became yellowish white after 1 week. Then in the following 3 weeks, the anthers began to swell and produce a mass of calluses (Fig. 1A). The induction frequency of calluses increased with the increasing of 2,4-D concentration from 0.05 mM up to 2.26 mM. When 2,4-D
Fig. 1. Embryogenesis in cultured anthers of loquat (E. japonica L. cv. ‘Dawuxing’). Panel (A): Calluses bursting open anther wall and growing out (the arrowhead indicates the anther wall) (bar = 2.0 mm). Panel (B): Embryogenic calluses with proembryos (the arrowhead indicates the protuberant structures with smooth surfaces, bar = 2.0 mm). Panel (C): A cluster of embryos at the globular stage (bar = 0.5 mm). Panel (D): A cluster of embryos at different developmental stages (the arrowheads indicate the cotyledonary embryos) (bar = 2.0 mm). Panel (E): Histological observation of globular and heart-shape embryos (arrowheads) (bar = 0.5 mm). Panel (F): Histological observation of a cotyledonary embryo with meristematic region (arrowhead) (bar = 1.0 mm).
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concentration increased to 4.52 mM, callus induction rate of cv. ‘Longquan1’ showed a sign of decline, while cv. ‘Dawuxing’ and ‘Zaozhong6’ showed a significant decline.
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were attached loosely to the mother calluses with a short suspensor-like structure at the basal end and occasionally got detached from parent calluses and continued their further development.
3.2. Embryo differentiation Calluses induced from 2-day cold pretreated anthers on MS medium supplemented with 7% (w/v) sucrose, 0.6% (w/v) agar, 2.26 mM 2,4-D and 8.90 mM BA were used to achieve embryo differentiation. These calluses were separated from anther wall and transferred to MS medium added different concentrations and combinations of ZT, IBA and NAA for induction of embryogenesis. When transferred to media devoid of growth regulators, calluses gradually became necrotic after 4 weeks. On the media containing 0.05 mM of ZT, multicellular structures (protuberant structures with coarse surfaces) and proembryos (protuberant structures with smooth surfaces) were observed under stereoscopic microscope. Even after prolonged maintenance or subcultured on the same medium, these calluses failed to produce embryos, and the multicellular structures and proembryos did not showed further development. On the media containing 0.46 mM of ZT, embryogenesis did not occur, and the calluses continued growing but no protubertant structure was formed. Among the three varieties used in the investigation, only cv. ‘Dawuxing’ underwent embryogenesis on two treatments in which 0.23 mM ZT were used (Table 2). Initially, the differentiated calluses became embryogenic and showed a high degree of friability with many globular structures (Fig. 1B). Subsequently, the embryogenic calluses developed into globular (Fig. 1C and E), heart (Fig. 1E), torpedo and cotyledonary stage (Fig. 1D and F) of embryos. After 4 weeks of subculture, the embryogenic calluses differentiated into clumps of embryos (Fig. 1C and D) on the medium containing MS basal salts supplemented with 0.23 mM ZT, 0.11 or 0.21 mM NAA, and 0.10 or 0.20 mM IBA. It was frequently observed that all the embryos in cluster did not develop synchronously (Fig. 1D), i.e., the embryos were found to be in different stages of development. A few embryos developed more quickly and probably suppressed the growth of other embryos. The rapidly growing cotyledonary embryos
3.3. Maturation, germination and plant conversion of embryos In all the treatment, the embryos increased in size during 4 weeks of culture on maturation media. After 4 weeks, embryos were transferred to germination medium and the germination rates varied significantly with the previous maturation treatments (Table 3). The frequency of embryos with only root development was highest in the treatments supplemented with 3% sucrose at 25 8C. Plantlet conversion rate was significantly enhanced in embryos matured on media supplemented with 3% or 5% sucrose at 4 8C (Fig. 2A and B). After another 4 weeks on the germination medium, complete plantlets were obtained (Fig. 2C). The effect of the maturation treatment on embryos with only shoot development (Table 3) was not significant, with the best results occurring on the media with 5% sucrose at 4 8C (10.5%). Considering the percentage of embryos developing into plantlets, the best results were achieved in the cold treatment (4 8C) on the media supplemented with 3% or 5% sucrose, giving a total of 72.5% and 52.5% of matured embryos eventually growing into normal plants, respectively. 3.4. Transplantation and cytological analysis Regenerated plants bearing six to seven leaves were taken out of the culture vessels and washed thoroughly in distilled water and transplanted in plastic cups containing a mixture of autoclaved peat, perlite and garden soil (1:1:1). Hardening of plotted plants for 8 weeks in a growth chamber was found essential. Of 155 plantlets transferred, 132 survived (Fig. 2D). Fifteen to twenty root tip cells were analyzed by counting chromosome numbers to determine the ploidy level of the regenerants. Out of 30 regenerants tested, 26 plantlets were haploids (n = 17) (Fig. 2E) and the remaining 4 plantlets were diploids (Fig. 2F).
Table 2 Effect of ZT, NAA and IBA on embryo differentiation from anther-derived calluses of cv. ‘Dawuxing’ ZT
NAA + IBA
No. of calluses forming embryos (%)
0.05 0.05 0.05 0.23 0.23 0.23 0.46 0.46 0.46
0.05 + 0.05 0.11 + 0.10 0.21 + 0.20 0.05 + 0.05 0.11 + 0.10 0.21 + 0.20 0.05 + 0.05 0.11 + 0.10 0.21 + 0.20
0 0 0 1 3 0 0 0 0
a
c c c (3.33) b (10.00) a c c c c
No. of embryos at different development stages per callusa Globular
Heart
Torpedo
Cotyledonary
0c 0c 0c 10.67 b 12.67 a 0c 0c 0c 0c
0b 0b 0b 8.33 a 8.11 a 0b 0b 0b 0b
0b 0b 0b 3.33 a 3.78 a 0b 0b 0b 0b
0b 0b 0b 8.00 a 10.67 a 0b 0b 0b 0b
Means having the same letter in a column were not significantly different according to Duncan’s multiple range test at P = 0.05.
Total number of embryos 0 0 0 91 317 0 0 0 0
c c c b a c c c c
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Table 3 Effect of various maturation treatments on subsequent germination of maturated embryos exhibiting shoot development, root development or plantlet conversion Temperature (8C)
4 4 25 25 a
Sucrose concentration (%)
3 5 3 5
Responsea Root development
Shoot development
Plantlet conversion
No.
%
No.
%
No.
%
1.5 2.0 5.5 4.5
7.5 c 10 bc 27.5 a 22.5 ab
2.0 3.0 1.0 1.0
10 15 5 5
14.5 10.5 0.5 0
72.5 a 52.5 b 2.5 c 0c
Means having the same letter in the columns were not significantly different according to Duncan’s multiple range test (P = 0.05).
Fig. 2. Germination and plantlet conversion of maturated embryos. Panel (A): A germinating embryo (bar = 2 mm). Panel (B): Plant conversion (bar = 2 mm). Panel (C): Regeneration of the whole plantlets in the conical flask (bar = 2 cm). Panel (D): A successfully transplanted whole plant (bar = 4 cm). Panel (E): Chromosomes of root tip cells of regenerated haploid plantlet (n = 17) (bar = 20 mm). Panel (F): Chromosomes of root tip cells of regenerated diploid plantlet (2n = 2x = 34) (bar = 20 mm).
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4. Discussion Pretreatment, culture medium composition and culture environment influence the efficiency of callus induction and embryo production (Sopory and Munshi, 1996). Shock treatment of anthers or whole flowers, including high or low temperatures for various lengths of time prior to culture, has been applied to a variety of plant species (Immonen and Robinson, 2000). Cold pretreatment is easy to practise and has been used to induce embryogenesis from isolated anthers as it disrupts the cytoskeleton in microspores in the initial phase (Ferrie et al., 1995). Application of cold pretreatment has become an essential measure to increase the efficiency of androgenesis in many species (Touraev et al., 1997; Pechan and Smykal, 2001). In our study, we found that in loquat the cold pretreatment of flower buds was necessary for subsequent callus induction from anthers, and 48 h of cold (4 8C) pretreatment proved to be optimal as it gave the best callus induction. However, the callusing potential of loquat anthers was impaired by prolonged cold pretreatment. When the cold pretreatment was up to 144 h, the anthers turned brown a few minutes after being removed from the buds. Early anther browning was associated with low callus induction rate, and the anthers became necrotic within 1 week. Therefore, cold pretreatment was one of key factors for callus induction in loquat anther culture. The presence of an appropriate concentration of PGRs in the medium plays a critical role in callus or embryo formation in anther culture (Sopory and Munshi, 1996). In present study, there was no sign of callus formation when anthers were cultured either on basal MS medium or on MS medium supplemented with IBA, NAA, KT, BA, and TDZ even after 8 weeks of culture, and the anthers subsequently became necrotic (data were not presented). Callus induction was significantly influenced by the concentration of 2,4-D. Medium without 2,4D was invalid for callus induction, which suggested that 2,4-D might be absolutely necessary for the dedifferentiation of loquat anthers. Calluses induced from 2-day cold pretreated anthers on MS medium supplemented with 7% (w/v) sucrose, 0.6% (w/v) agar, 2.26 mM 2,4-D and 8.90 mM BA eventually achieved embryo differentiation. The original anther culture experiments on Datura involved the inclusion of auxin and cytokinin in the medium (Guha and Maheshwari, 1964). In most cases, anthers are plated on culture media in which variations of type, concentration and combination of PGRs are presented as essential experimental factors (Assani et al., 2003; Chaturvedi et al., 2003). In the present study, to enhance the embryogenic potential, antherderived calluses were transferred to media with different concentrations and combinations of auxins and cytokinins. However, in almost all treatment, calluses failed to differentiate embryos, and the multicellular structures or proembryos showed no further development. Further growth could only proceed when the multicellular structures or proembryos underwent embryogenesis. Calluses cultured on the medium containing 3% (w/v) sucrose, 0.23 mM ZT, 0.11 mM NAA and 0.10 mM IBA resulted in the highest embryo differentiation rate
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(10.0%). The result of histological study showed that the embryos developed through the typical globular, torpedo, heart and cotyledon stage. Differentiation did not occur when the calluses were cultured on media devoid of any growth regulators, and finally the calluses turned brown and became necrotic. Therefore, it seemed that exogenous growth regulator supply was essential for induction of embryogenesis and a particular combination of cytokinins with auxins would be necessary. It appears to be more essential to select genotypes that are best able to regenerate plants than those which produce a greater number of calluses (Sopory and Munshi, 1996). In the present study, the low frequency of embryogenesis induction on media with different PGRs combinations may suggest the importance of genotype of the donor plant which might play a more decisive role in the induction of embryogenesis than the externally supplied growth regulators. The three loquat cultivars used in the study varied in embryo differentiation. Only cv. ‘Dawuxing’ underwent embryogenesis and formed a cluster of embryos. Embryo differentiation did not occur in cv. ‘Longquan1’ and ‘Zaozhong6’. Although the multicellular structures and proembryos were formed, they showed no further development and failed to produce embryos in cv. ‘Longquan1’ and ‘Zaozhong6’. Therefore, we could draw a conclusion that the genotype would play a more important role for embryogenesis than PGRs in anther culture of loquat. Generally, the objective of cold treatment is to break the dormancy imposed by ABA, promote germination and synchronize shoot and root development (Merkle et al., 1995). In the present investigation, chilling at 4 8C enhanced the recovery of plantlet from matured embryos: 72.5% or 52.5% of embryos directly converting into plantlets, contrasting to the poor responses of 2.5% or 0%, respectively at 25 8C. Obviously, the application of a cold-treatment for embryo maturation was effective for achieving the subsequent plant conversion. The result of cytological analysis showed that out of 30 regenerants tested, 26 plantlets were haploids (n = 17) and the remaining 4 plantlets were diploids (n = 34). The possible interpretation of the 4 diploid plantlets would be that they originated from anther walls, or the microspores spontaneously undergoing chromosome doubling during in vitro culture. Thus, we could draw a conclusion that the 26 regenerants were derived from microspores and haploid production can be realized through anther culture in loquat. Acknowledgments We acknowledge partial financial support to the research from Chinese Ministry of Agriculture and Sichuan Department of Science and Technology. We are grateful to Prof. J. Moorby, Dr. R. Fordham, Prof. Z. Ren, Prof. Y. Zheng and Prof. G. Liang for their critical comments on the manuscript. We thank Dr. P.G. Luo and Dr. X. Wan for providing technical assistance. The authors also wish to thank the reviewers and editors for their valuable comments on the original manuscript.
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Guha, S., Maheshwari, S.C., 1964. In vitro production of embryos from anthers of Datura. Nature (Lond.) 204, 497. Guha, S., Maheshwari, S.C., 1966. Cell division and differentiation of embryos in the pollen grains of Datura in vitro. Nature (Lond.) 212, 97–98. Immonen, S., Robinson, J., 2000. Stress treatments and ficoll for improving green plant regeneration in triticale anther culture. Plant Sci. 150, 77–84. Merkle, S.A., Parrot, W.A., Flinn, B.S., 1995. Morphogenic aspects of embryogenesis. In: Thorpe, T.A. (Ed.), In vitro Embryogenesis in Plants. Kluwer Academic Publishers, Dordrecht, pp. 155–203. Murashige, T., Skoog, F.G., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473–479. Pechan, P.M., Smykal, P., 2001. Androgenesis: affecting the fate of the male gametophyte. Physiol. Plant. 111, 1–8. Radojevic, L., Marinkovic, N., Jevremovic, S., 2000. Influence of the sex of flowers on androgenesis in Aesculus hippocastanum L. anther culture. In Vitro Cell Dev. Plant. 36, 464–469. Rimberia, F.K., Sungagawa, H., Urasaki, N., Ishimine, Y., Adaniya, S., 2005. Embryo induction via anther culture in papaya and sex analysis of the derived plantlets. Sci. Hort. 103, 199–208. Sopory, S.K., Munshi, M., 1996. Anther culture. In: Jain, S.M., Sopory, S.K., Veilleux, R.E. (Eds.), In vitro Haploid Production in Higher Plants, vol. I. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 145–176. Touraev, A., Vicente, O., Heberle-Bors, E., 1997. Initiation of embryogenesis by stress. Trends Plant Sci. 2, 297–302. Vilanova, S., Badenes, M.L., Martines-Calvo, J., Lla, G., 2001. Analysis of germplasm (Eriobotrya japonica Lindl.) by RAPD molecular markers. Euphytica 121, 25–29.
Scientia Horticulturae 115 (2008) 329–336 www.elsevier.com/locate/scihorti
Embryogenesis and plant regeneration from anther culture in loquat (Eriobotrya japonica L.) Li Junqianga,b, Wang Yongqinga,*, Lin Lihuab, Zhou Lijunb, Luo Nana, Deng Qunxiana, Xian Junrena, Hou Chunxiaa, Qiu Yuana a
College of Forestry and Horticulture, Sichuan Agricultural University, Ya’an 625014, Sichuan, China Department of Bioengineering, Yibin Vocational & Technical College, Yibin 644003, Sichuan, China
b
Received 24 October 2006; received in revised form 30 September 2007; accepted 4 October 2007
Abstract The object of this study was to induce embryogenesis and establish plant regeneration system for anther culture in loquat (Eriobotrya japonica L.). Cold pretreatment was a key factor, and supplement of 2,4-D in the media was absolutely necessary for induction of calluses from cultured loquat anthers. The best response of anthers to in vitro culture was obtained when a 48-h cold pretreatment was employed to flower buds at 4 8C in darkness. Genotype was a decisive factor for embryo differentiation. When anther-derived calluses of three loquat cultivars, i.e., cv. ‘Longquan1’, ‘Dawuxing’ and ‘Zaozhong6’, were transferred to embryo differentiation medium, embryos were induced only for cv. ‘Dawuxing’ on MS medium containing 3% sucrose, 0.23 mM ZT in combination with 0.05 mM NAA + 0.05 mM IBA or 0.11 mM NAA + 0.10 mM IBA, and the differentiation rates were 3.33% and 10.00%, respectively. The results of histological studies showed that embryos developed through typical globular, heart, torpedo and cotyledon stages after 4 weeks of culture. The treatment designed to mature the embryos on medium containing 3% of sucrose at 4 8C under darkness for 4 weeks was effective for subsequent embryo germination and plant conversion, which gave rise to 72.5% plant recovery. Cytological studies showed that 26 plantlets were haploids (n = 17) and the remaining 4 plantlets were diploids for the 30 regenerants tested. # 2007 Elsevier B.V. All rights reserved. Keywords: Anther culture; Callus induction; Embryogenesis; Eriobotrya japonica L.; Haploid; Plant regeneration
1. Introduction Plant breeders have always been interested in shortening the period required for breeding. But in traditional breeding systems, more than six generations are needed for homogenizing alleles for a particular trait (Rimberia et al., 2005). At haploid level, every gene is hemizygous. After chromosome doubling, which makes an identical copy of each haploid chromosome, every genotype becomes homozygous (Baenziger, 1996). Thus, production of haploids offers new possibilities for genetic studies and for an increased efficiency in selection. Anther culture, originally developed by Guha and Maheshwari (1964, 1966), allows rapid production of haploids, and
Abbreviations: BA, benzyladenine; 2,4-D, 2,4-dichlorophenoxyacetic acid; IBA, indolebutyric acid; KT, kinetin; MS medium, Murashige and Skoog medium (1962); NAA, a-naphthaleneacetic acid; PGRs, plant growth regulators; TDZ, thidiazuron; ZT, zeatin. * Corresponding author. Tel.: +86 835 2882782; fax: +86 835 2882578. E-mail address: [email protected] (Y. Wang). 0304-4238/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2007.10.007
subsequently chromosome doubling can lead to completely homozygous diploid plants. This technique has become one of the major sources of haploid plant production used to develop homozygous diploids in plant breeding programs (Bajaj, 1983). Compared with conventional inbreeding, the in vitro androgenesis technique enables a faster generation of virtually fully homozygous lines (Aulinger et al., 2003). Androgenesis is a valuable procedure for obtaining haploids and doubled haploids in selection and breeding programs, especially for woody species that are heterozygous (Radojevic et al., 2000). Nevertheless, there are a number of factors that affect androgenesis, including genotype and physiological state of the donor plant, anther age and pollen developmental stage, pre-culture treatment, physical factor (temperature, light, atmospheric condition, anther density and orientation and pH) and chemical factor (culture medium, sugar and plant growth regulator) (Sopory and Munshi, 1996). As a result, so far, there have been only a limited number of successes of anther culture in woody species. As an important economic fruit crop, loquat (Eriobotrya japonica L.) is widely cultivated between 208 and 358 latitude
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including China, Japan, India, Pakistan, Madagascar, Reunion Island, Mauritius Island, the Mediterranean countries (Spain, Turkey, Italy, Greece, Israel), the United States (mainly California and Florida), Brazil, Venezuela, and Australia (Badenes et al., 2000; Vilanova et al., 2001). This crop is characterized by a long reproductive cycle and juvenile phase (both of several years). Because of heterozygosity and long juvenile period of the plant, only a limited number of genetic studies have been performed and no long-term breeding program has ever been established for this crop. The traditional breeding methods are time-consuming and limited by space required for field experiments. Consequently, there is a need to shorten the breeding period using techniques such as haploid induction via anther culture. The present paper reports, for the first time, the establishment of plant regeneration system and haploid production through embryogenesis from anther culture of loquat. 2. Materials and methods
combination with a-naphthaleneacetic acid (NAA) + indolebutyric acid (IBA) (0.05 + 0.05, 0.11 + 0.10 and 0.21 + 0.20 mM). The conical flasks were capped with non-absorbent cotton plugs wrapped with one layer of cheesecloth. The media were adjusted to pH 5.6 prior to addition of agar and sterilized at 122 8C and 104 kPa pressure for 17 min. For callus induction, cultures were maintained at 25 1 8C in the dark. For embryo differentiation, cultures were cultured in a culture chamber at 25 1 8C under 16 h daily illumination with 2000 lx fluorescent light. 2.2. Histological study Embryos in different developmental stages were fixed in FAA (70% ethanol:formalin:acetic acid, 18:1:1, v/v/v), rinsed, dehydrated and embedded in paraffin (56–58 8C) using classical techniques. Sections (10 mm thick) were cut using a rotary microtome, and stained with haematoxylin. Then, sections were observed under a light microscope and documented by a digital camera (Motic BA200).
2.1. In vitro culture of anthers 2.3. Embryo maturation, germination and plant conversion Anthers of loquat (E. japonica L.) cv. ‘Longquan1’, ‘Dawuxing’ and ‘Zaozhong6’ were the experimental materials in the present investigations. The mother plants were grown in the experimental plots using standard agronomic practices. Flower buds (5–6 mm in diameter) were harvested, which contained approximately 80% uninucleate microspores confirmed by periodically microscopically checking the microspore stage. The flower buds were collected in a conical flask with a piece of wet cotton to maintain a high humidity and kept at 4 8C in dark for 0 (control), 24, 48, 96 and 144 h. Before culturing of anthers, the thick floss of the sepals was completely scraped off (otherwise they would make it difficult to sterilize the flower buds) with a surgical scalpel and then washed in distilled water containing 3% (v/v) Tween (detergent) for 5 min. The final step of sterilization and further operations were carried out in a laminar air-flow cabinet under aseptic conditions. The flower buds were surface-sterilized by immersion in 0.1% (w/v) mercuric chloride solution with periodic agitation for 8 min, and washed with sterile distilled water for five times. After removing filament, the intact anthers were plated horizontally, with the connective tissues contacting the medium, in the conical flasks (150 ml) containing 30 ml of medium. Callus induction medium consisted of Murashige and Skoog (MS) (1962) mineral salts and vitamins, 7% (w/v) sucrose, 0.6% (w/v) agar, and it was supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) (0.05, 0.09, 0.23, 0.45, 1.36, 2.26 and 4.52 mM) and benzyladenine (BA) (8.90 mM). Calluses which were derived from MS medium containing 7% (w/v) sucrose, 0.6% (w/v) agar, 2.26 mM 2,4-D and 8.90 mM BA (a 2-day cold pretreatment was employed to these cultured anthers) were used to achieve embryo differentiation. The successfully induced calluses were separated from anther walls and cultured on an embryo differentiation medium. Embryo differentiation medium consisted of MS mineral salts and vitamins, 3% (w/v) sucrose, 0.6% (w/v) agar, and zeatin (ZT) (0.05, 0.23 and 0.46 mM) in
The effect of various maturation treatments was evaluated in terms of subsequent embryo germination and plantlet conversion ability. White opaque cotyledonary embryos were isolated from embryo clusters and cultured on different maturation media, consisting of PGR-free basal medium supplemented with sucrose (3% or 5%), and stored at 4 or 25 8C for 4 weeks under darkness. After 4 weeks of culture on maturation medium, embryos were transferred to MS basal medium with 3% sucrose. And then, they were transferred into Petri dishes containing 20 ml germination medium (basal MS medium) for 4 weeks. Photos of callus induction, embryo differentiation, embryo maturation, embryo germination, plantlet conversion were taken using a stereoscopic microscope (Motic DM143) and a digital camera (SONY F717). 2.4. Transplantation of plantlets and cytological studies Well-developed plantlets were removed from culture vessels and washed in distilled water to remove traces of the medium. These plantlets were then transplanted to plastic cups containing a mixture of autoclaved peat, perlite and garden soil (1:1:1). The plantlets were grown in a growth chamber (25 1 8C and 16 h photoperiod of 2000 lx illumination, 90% relative humidity). After 8 weeks, these plants were transferred to the greenhouse for further growth. For cytological studies, the root tips of 30 randomly selected regenerants were harvested and treated with 2 mM aqueous solution of 8-hydroxyquinoline at room temperature for 4 h. The pretreated root tips were fixed in acetic alcohol solution (3 parts of ethanol:1 part of glacial acetic acid) for 24 h. Subsequently root tips were washed in distilled water and hydrolyzed in 1N HCl at 60 8C for 10 min and stained with Feulgen reagent for 1 h and squashed in 45% acetic acid (Armstrong, 1996). Chromosome preparations were photographed using a light microscope (Motic BA200).
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twice. Cultures were observed periodically and morphological changes were recorded at regular intervals of 4 weeks. The term of maturation in this study denotes the development of globular and heart-shaped embryos into distinct cotyledonary form which is a bipolar embryo with well-developed cotyledons, and the term germination means the development of shoots or roots or both shoots and roots (plant conversion). The results presented in this study are the mean of 300 anthers, 30 anther-derived calluses or 20 embryos per experiment. The data were subject to analysis of variance (ANOVA) to assess treatment differences and interactions using the SPSS statistical package for windows (release 11.0, SPSS INC). Significance between means was tested by Duncan’s multiple range test.
2.5. Data collection and analysis A factorial design was employed for callus induction experiments and a randomized complete block design was used for the other experiments. For callus induction, 300 anthers were cultured (20 anthers per conical flask and 15 replicates per treatment). For embryo differentiation, 30 calluses were cultured (10 calluses per conical flask and 3 replicates per treatment). For embryo maturation and germination experiments, each treatment was applied to 20 embryos (10 embryos per conical flask and 2 replicates per treatment). Callus induction and embryo differentiation experiments were repeated three times, and embryo maturation and germination experiments were repeated
Table 1 Effect of cold pretreatment to flower buds and PGRs on the callus induction of anthers after 4 weeks of culture in three loquat cultivars Cold pretreatment (h)
2,4-D (mM)
Anthers forming callusesa cv. ‘Longquan1’
cv. ‘Dawuxing’
cv. ‘Zaozhong6’
No.
No.
No.
%
0
0.05 0.09 0.23 0.45 1.36 2.26 4.52
0 11 0 0 2 0 5
0 3.67 0 0 0.67 0 1.67
24
0.05 0.09 0.23 0.45 1.36 2.26 4.52
15 31 55 68 77 88 85
5.00 10.33 18.33 22.67 25.67 29.33 28.33
48
0.05 0.09 0.23 0.45 1.36 2.26 4.52
20 34 55 58 37 208 168
96
0.05 0.09 0.23 0.45 1.36 2.26 4.52
114
0.05 0.09 0.23 0.45 1.36 2.26 4.52
Significance of two-way ANOVA 2,4-D concentration (A) Cold pretreatment (B) AB a **
%
5 0 3 0 3 0 0
1.67 0 1.00 0 1.00 0 0
F E D C B A A
18 33 34 37 47 46 45
6.00 11.00 11.33 12.33 15.67 15.33 15.00
6.67 11.33 18.33 19.33 12.33 69.33 56.00
E D C C D A B
23 39 55 55 52 169 151
3 7 15 20 32 53 45
1.00 2.33 5.00 6.67 10.67 17.67 15.00
E DE CD C B A A
1 5 6 12 16 27 22
0.33 1.67 2.00 4.00 5.33 9.00 7.33
C C C B B A A
%
3 0 6 0 2 0 1
1.00 0 2.00 0 0.67 0 0.33
C B B B A A A
38 37 52 53 61 72 69
12.67 12.33 17.33 17.67 20.33 24.00 23.00
D D C C BC A AB
7.67 13.00 18.33 18.33 17.33 56.33 50.33
E D C C C A B
44 59 73 103 79 256 226
14.67 19.67 24.33 34.33 26.33 85.33 75.33
F E D C D A B
2 2 7 22 53 61 53
0.67 0.67 2.33 7.33 17.67 20.33 17.67
D D D C B A B
9 18 25 41 59 132 127
3.00 6.00 8.33 13.67 19.67 44.00 42.33
E DE D C B A A
2 7 8 9 17 23 22
0.67 2.33 2.67 3.00 5.67 7.67 7.33
20 39 39 83 81 95 92
6.67 13.00 13.00 27.67 27.00 31.67 30.67
D C C B B A A
D CD C C B A AB
**
**
**
**
**
**
**
**
**
Means having the same letter in the columns were not significantly different according to Duncan’s multiple range test (P = 0.01). Significant at P = 0.01.
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3. Results 3.1. Effect of cold pretreatment of flower buds and PGRs on callus formation of cultured anthers The results of callus induction experiments were summarized in Table 1. Analysis of variance of the callus induction rate indicated that callus formation was significantly affected by cold pretreatment (P = 0.01), 2,4-D concentration (P = 0.01), and their interaction (P = 0.01). If the anthers were not coldtreated, callus induction rates were not significantly affected by 2,4-D concentration (P = 0.05). A 48-h cold shock was beneficial as it significantly increased the induction rate of
calluses, reached the apex of 69.33%, 56.33% and 85.33% for cv. ‘Longquan1’, ‘Dawuxing’ and ‘Zaozhong6’, respectively. Prolonged cold shock (96–144 h) dramatically decreased the potential of anthers to produce calluses because it increased the frequency of anthers to turn brown and then became necrotic. Regardless of the time of cold pretreatment used to the flower buds, the best results in terms of callus induction were obtained with the higher concentration of 2,4-D (2.26 mM). The responding anthers grew larger in size and became yellowish white after 1 week. Then in the following 3 weeks, the anthers began to swell and produce a mass of calluses (Fig. 1A). The induction frequency of calluses increased with the increasing of 2,4-D concentration from 0.05 mM up to 2.26 mM. When 2,4-D
Fig. 1. Embryogenesis in cultured anthers of loquat (E. japonica L. cv. ‘Dawuxing’). Panel (A): Calluses bursting open anther wall and growing out (the arrowhead indicates the anther wall) (bar = 2.0 mm). Panel (B): Embryogenic calluses with proembryos (the arrowhead indicates the protuberant structures with smooth surfaces, bar = 2.0 mm). Panel (C): A cluster of embryos at the globular stage (bar = 0.5 mm). Panel (D): A cluster of embryos at different developmental stages (the arrowheads indicate the cotyledonary embryos) (bar = 2.0 mm). Panel (E): Histological observation of globular and heart-shape embryos (arrowheads) (bar = 0.5 mm). Panel (F): Histological observation of a cotyledonary embryo with meristematic region (arrowhead) (bar = 1.0 mm).
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concentration increased to 4.52 mM, callus induction rate of cv. ‘Longquan1’ showed a sign of decline, while cv. ‘Dawuxing’ and ‘Zaozhong6’ showed a significant decline.
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were attached loosely to the mother calluses with a short suspensor-like structure at the basal end and occasionally got detached from parent calluses and continued their further development.
3.2. Embryo differentiation Calluses induced from 2-day cold pretreated anthers on MS medium supplemented with 7% (w/v) sucrose, 0.6% (w/v) agar, 2.26 mM 2,4-D and 8.90 mM BA were used to achieve embryo differentiation. These calluses were separated from anther wall and transferred to MS medium added different concentrations and combinations of ZT, IBA and NAA for induction of embryogenesis. When transferred to media devoid of growth regulators, calluses gradually became necrotic after 4 weeks. On the media containing 0.05 mM of ZT, multicellular structures (protuberant structures with coarse surfaces) and proembryos (protuberant structures with smooth surfaces) were observed under stereoscopic microscope. Even after prolonged maintenance or subcultured on the same medium, these calluses failed to produce embryos, and the multicellular structures and proembryos did not showed further development. On the media containing 0.46 mM of ZT, embryogenesis did not occur, and the calluses continued growing but no protubertant structure was formed. Among the three varieties used in the investigation, only cv. ‘Dawuxing’ underwent embryogenesis on two treatments in which 0.23 mM ZT were used (Table 2). Initially, the differentiated calluses became embryogenic and showed a high degree of friability with many globular structures (Fig. 1B). Subsequently, the embryogenic calluses developed into globular (Fig. 1C and E), heart (Fig. 1E), torpedo and cotyledonary stage (Fig. 1D and F) of embryos. After 4 weeks of subculture, the embryogenic calluses differentiated into clumps of embryos (Fig. 1C and D) on the medium containing MS basal salts supplemented with 0.23 mM ZT, 0.11 or 0.21 mM NAA, and 0.10 or 0.20 mM IBA. It was frequently observed that all the embryos in cluster did not develop synchronously (Fig. 1D), i.e., the embryos were found to be in different stages of development. A few embryos developed more quickly and probably suppressed the growth of other embryos. The rapidly growing cotyledonary embryos
3.3. Maturation, germination and plant conversion of embryos In all the treatment, the embryos increased in size during 4 weeks of culture on maturation media. After 4 weeks, embryos were transferred to germination medium and the germination rates varied significantly with the previous maturation treatments (Table 3). The frequency of embryos with only root development was highest in the treatments supplemented with 3% sucrose at 25 8C. Plantlet conversion rate was significantly enhanced in embryos matured on media supplemented with 3% or 5% sucrose at 4 8C (Fig. 2A and B). After another 4 weeks on the germination medium, complete plantlets were obtained (Fig. 2C). The effect of the maturation treatment on embryos with only shoot development (Table 3) was not significant, with the best results occurring on the media with 5% sucrose at 4 8C (10.5%). Considering the percentage of embryos developing into plantlets, the best results were achieved in the cold treatment (4 8C) on the media supplemented with 3% or 5% sucrose, giving a total of 72.5% and 52.5% of matured embryos eventually growing into normal plants, respectively. 3.4. Transplantation and cytological analysis Regenerated plants bearing six to seven leaves were taken out of the culture vessels and washed thoroughly in distilled water and transplanted in plastic cups containing a mixture of autoclaved peat, perlite and garden soil (1:1:1). Hardening of plotted plants for 8 weeks in a growth chamber was found essential. Of 155 plantlets transferred, 132 survived (Fig. 2D). Fifteen to twenty root tip cells were analyzed by counting chromosome numbers to determine the ploidy level of the regenerants. Out of 30 regenerants tested, 26 plantlets were haploids (n = 17) (Fig. 2E) and the remaining 4 plantlets were diploids (Fig. 2F).
Table 2 Effect of ZT, NAA and IBA on embryo differentiation from anther-derived calluses of cv. ‘Dawuxing’ ZT
NAA + IBA
No. of calluses forming embryos (%)
0.05 0.05 0.05 0.23 0.23 0.23 0.46 0.46 0.46
0.05 + 0.05 0.11 + 0.10 0.21 + 0.20 0.05 + 0.05 0.11 + 0.10 0.21 + 0.20 0.05 + 0.05 0.11 + 0.10 0.21 + 0.20
0 0 0 1 3 0 0 0 0
a
c c c (3.33) b (10.00) a c c c c
No. of embryos at different development stages per callusa Globular
Heart
Torpedo
Cotyledonary
0c 0c 0c 10.67 b 12.67 a 0c 0c 0c 0c
0b 0b 0b 8.33 a 8.11 a 0b 0b 0b 0b
0b 0b 0b 3.33 a 3.78 a 0b 0b 0b 0b
0b 0b 0b 8.00 a 10.67 a 0b 0b 0b 0b
Means having the same letter in a column were not significantly different according to Duncan’s multiple range test at P = 0.05.
Total number of embryos 0 0 0 91 317 0 0 0 0
c c c b a c c c c
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Table 3 Effect of various maturation treatments on subsequent germination of maturated embryos exhibiting shoot development, root development or plantlet conversion Temperature (8C)
4 4 25 25 a
Sucrose concentration (%)
3 5 3 5
Responsea Root development
Shoot development
Plantlet conversion
No.
%
No.
%
No.
%
1.5 2.0 5.5 4.5
7.5 c 10 bc 27.5 a 22.5 ab
2.0 3.0 1.0 1.0
10 15 5 5
14.5 10.5 0.5 0
72.5 a 52.5 b 2.5 c 0c
Means having the same letter in the columns were not significantly different according to Duncan’s multiple range test (P = 0.05).
Fig. 2. Germination and plantlet conversion of maturated embryos. Panel (A): A germinating embryo (bar = 2 mm). Panel (B): Plant conversion (bar = 2 mm). Panel (C): Regeneration of the whole plantlets in the conical flask (bar = 2 cm). Panel (D): A successfully transplanted whole plant (bar = 4 cm). Panel (E): Chromosomes of root tip cells of regenerated haploid plantlet (n = 17) (bar = 20 mm). Panel (F): Chromosomes of root tip cells of regenerated diploid plantlet (2n = 2x = 34) (bar = 20 mm).
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4. Discussion Pretreatment, culture medium composition and culture environment influence the efficiency of callus induction and embryo production (Sopory and Munshi, 1996). Shock treatment of anthers or whole flowers, including high or low temperatures for various lengths of time prior to culture, has been applied to a variety of plant species (Immonen and Robinson, 2000). Cold pretreatment is easy to practise and has been used to induce embryogenesis from isolated anthers as it disrupts the cytoskeleton in microspores in the initial phase (Ferrie et al., 1995). Application of cold pretreatment has become an essential measure to increase the efficiency of androgenesis in many species (Touraev et al., 1997; Pechan and Smykal, 2001). In our study, we found that in loquat the cold pretreatment of flower buds was necessary for subsequent callus induction from anthers, and 48 h of cold (4 8C) pretreatment proved to be optimal as it gave the best callus induction. However, the callusing potential of loquat anthers was impaired by prolonged cold pretreatment. When the cold pretreatment was up to 144 h, the anthers turned brown a few minutes after being removed from the buds. Early anther browning was associated with low callus induction rate, and the anthers became necrotic within 1 week. Therefore, cold pretreatment was one of key factors for callus induction in loquat anther culture. The presence of an appropriate concentration of PGRs in the medium plays a critical role in callus or embryo formation in anther culture (Sopory and Munshi, 1996). In present study, there was no sign of callus formation when anthers were cultured either on basal MS medium or on MS medium supplemented with IBA, NAA, KT, BA, and TDZ even after 8 weeks of culture, and the anthers subsequently became necrotic (data were not presented). Callus induction was significantly influenced by the concentration of 2,4-D. Medium without 2,4D was invalid for callus induction, which suggested that 2,4-D might be absolutely necessary for the dedifferentiation of loquat anthers. Calluses induced from 2-day cold pretreated anthers on MS medium supplemented with 7% (w/v) sucrose, 0.6% (w/v) agar, 2.26 mM 2,4-D and 8.90 mM BA eventually achieved embryo differentiation. The original anther culture experiments on Datura involved the inclusion of auxin and cytokinin in the medium (Guha and Maheshwari, 1964). In most cases, anthers are plated on culture media in which variations of type, concentration and combination of PGRs are presented as essential experimental factors (Assani et al., 2003; Chaturvedi et al., 2003). In the present study, to enhance the embryogenic potential, antherderived calluses were transferred to media with different concentrations and combinations of auxins and cytokinins. However, in almost all treatment, calluses failed to differentiate embryos, and the multicellular structures or proembryos showed no further development. Further growth could only proceed when the multicellular structures or proembryos underwent embryogenesis. Calluses cultured on the medium containing 3% (w/v) sucrose, 0.23 mM ZT, 0.11 mM NAA and 0.10 mM IBA resulted in the highest embryo differentiation rate
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(10.0%). The result of histological study showed that the embryos developed through the typical globular, torpedo, heart and cotyledon stage. Differentiation did not occur when the calluses were cultured on media devoid of any growth regulators, and finally the calluses turned brown and became necrotic. Therefore, it seemed that exogenous growth regulator supply was essential for induction of embryogenesis and a particular combination of cytokinins with auxins would be necessary. It appears to be more essential to select genotypes that are best able to regenerate plants than those which produce a greater number of calluses (Sopory and Munshi, 1996). In the present study, the low frequency of embryogenesis induction on media with different PGRs combinations may suggest the importance of genotype of the donor plant which might play a more decisive role in the induction of embryogenesis than the externally supplied growth regulators. The three loquat cultivars used in the study varied in embryo differentiation. Only cv. ‘Dawuxing’ underwent embryogenesis and formed a cluster of embryos. Embryo differentiation did not occur in cv. ‘Longquan1’ and ‘Zaozhong6’. Although the multicellular structures and proembryos were formed, they showed no further development and failed to produce embryos in cv. ‘Longquan1’ and ‘Zaozhong6’. Therefore, we could draw a conclusion that the genotype would play a more important role for embryogenesis than PGRs in anther culture of loquat. Generally, the objective of cold treatment is to break the dormancy imposed by ABA, promote germination and synchronize shoot and root development (Merkle et al., 1995). In the present investigation, chilling at 4 8C enhanced the recovery of plantlet from matured embryos: 72.5% or 52.5% of embryos directly converting into plantlets, contrasting to the poor responses of 2.5% or 0%, respectively at 25 8C. Obviously, the application of a cold-treatment for embryo maturation was effective for achieving the subsequent plant conversion. The result of cytological analysis showed that out of 30 regenerants tested, 26 plantlets were haploids (n = 17) and the remaining 4 plantlets were diploids (n = 34). The possible interpretation of the 4 diploid plantlets would be that they originated from anther walls, or the microspores spontaneously undergoing chromosome doubling during in vitro culture. Thus, we could draw a conclusion that the 26 regenerants were derived from microspores and haploid production can be realized through anther culture in loquat. Acknowledgments We acknowledge partial financial support to the research from Chinese Ministry of Agriculture and Sichuan Department of Science and Technology. We are grateful to Prof. J. Moorby, Dr. R. Fordham, Prof. Z. Ren, Prof. Y. Zheng and Prof. G. Liang for their critical comments on the manuscript. We thank Dr. P.G. Luo and Dr. X. Wan for providing technical assistance. The authors also wish to thank the reviewers and editors for their valuable comments on the original manuscript.
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