Development of synthetic seeds involving androgenic and pro-embryos in elite indica rice
Bidhan Roy*1 and Asit B Mandal
Biotechnology Section, Central Agricultural Research Institute, P O Box No. 181, Port Blair 744 105, India Received 12 February 2007; revised 19 February 2008; accepted 22 April 2008
Synthetic seeds were produced from anther-derived mass-multiplied embryos and pro-embryos of rice (Oryza sativa L.) var. IR 72. A high dose (4-6 mg L-1) of BAP was found to produce a large number of dormant embryos, pro-embryos and embryo-like structures in about 45 d. These were encapsulated in sodium alginate (2.5% w/v) matrix. Germination and plantlet regeneration capacity of the encapsulated seeds were tested by culturing them on MS fortified with different combinations and concentrations of BAP, Kn and NAA. The result indicated that BAP in combination with lower concentrations of NAA increased germination of beaded embryos over control (MS without hormones). High percent of germination (55-87.5%) was observed when MS was supplemented with BAP and lower concentration of NAA; whereas, addition of Kn in MS reduced the germination percentage. The germination of unbeaded pro-embryos was 92.5% on MS basal medium. The reduced rate of germination of artificial seeds may be attributed to the damage incurred while separating the embryos from clusters and/or owing to adverse effects of chemicals used for encapsulation. Moderate germination (40.0%) was seen on sterile sand. Synthetic seeds may be used for in vitro propagation as well as genetic transformation experiments, especially involving biolistics.
Keywords: Androgenic embryo, anther culture, artificial seeds, pro-embryo, rice, sodium alginate
Introduction
Production of synthetic seeds, endowed with high germination rate under in vitro and in vivo conditions, bears immense potential as an alternative of true seeds. It is an emerging area with great potential for large-scale production of propagules at lower cost with ease in handling and transport of crop plants1,2. Artificial seeds consisting of culture-derived embryos encased in a protective matrix were found to be germinable for mass production of elite varieties3. In several plants, conversion of artificial seeds into plantlets was reported4-7. However, information about production of artificial seeds from androgenic embryos derived from androgenic callus in rice is scanty. Initially, the preparation of artificial seeds was restricted to encapsulation of somatic embryos in a protective jelly and plantlet development from artificial seeds was observed in a wide variety of crops. The same principle has been exploited in the present study. However, the encapsulation of anther-
derived embryos is extremely limited. Haploid plant breeding has been well established as a tool for crop improvement8-12. The induction of pollen embryogenesis, which genetically differs from zygotic embryogenesis, may be used for this purpose.
The essential prerequisite for application of the artificial seed technology en masse involves large- scale production of high quality micro-propagules, which at present is a serious limiting factor1. Embryogenesis in androgenic calli in indica rice has been comparatively low than japonica and tropical japonica varieties. Owing to certain inherent problems in callus induction and plantlet regeneration, the rate of production of uniform and high quality embryos was found to be much lower. As a result, efficient and quality artificial seed production was found to be not much successful involving anther- derived callus in rice. To overcome this, appropriate protocol to produce a large number of embryos, pro- embryos and embryo-like structures from anther- derived plantlets has been developed for an elite indica rice var. IR 72. When androgenic shootlets were cultured on MS13 medium supplemented with 4 and 6 mg L-1 BAP14, they produced more than 100
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*Author for correspondence:
Tel: 91-3582-270587; Fax: 91-3582-270157 E-mail: bcroy10@yahoo.com
1Present address: Department of Genetics and Plant Breeding, Uttar Banga Krishi Viswavidyalaya, Pundibari, Cooch Behar 736 165, India
dormant embryos per culture within 45 d of culture.
These dormant embryos, pro-embryos and embryo- like structures produced shoots and roots en route embryogenesis on MS with no hormones, and were used as source materials for preparation of artificial seeds. The present communication deals with encapsulation of embryos, pro-embryos and embryos- like structures of androgenic origin and assessment of their germination in vitro and in vivo to ascertain the feasibility of their use as an alternative of true seeds, and possibility of use in genetic transformation experiment especially by using biolistic system.
Materials and Methods
Callus Induction and Plantlet Regeneration
'IR 72', an elite indica rice variety obtained from International Rice Research Institute (IRRI), Manila, Philippines, was grown in the experimental net-house of Central Agricultural Research Institute, Port Blair, India. For anther culture, the primary panicles enclosed in leaf sheaths were collected from the main tillers when the microspores were in mid-uninucleate to early bio-nucleate condition. The panicles were cold shocked for 8 d at 8°C in a BOD incubator prior to anther plating. On the day of culture, selected spikelets were surface sterilized in tissue culture bottles with 0.1% freshly prepared HgCl2 solution for 10 min. The HgCl2 was then drained off and the panicles were washed 4 times in sterile distilled water.
Anthers were plated aseptically onto radiation sterilized Petridishes having callus induction medium (CIM) containing N6 with 2 mg L-l 2,4-D, 6% sucrose and 0.8% agar. The cultures were sealed with parafilm and kept in complete dark at 25±2°C.
Embryogenic calli of at least ~ 2 mm diameter were transferred to culture tubes containing 10 mL of regeneration medium (RM) consisting of MS with 1 mg L-l BAP, 1 mg L-l Kn, 0.5 mg L-l NAA, 3%
(w/v) sucrose and 0.8% (w/v) agar. Inoculated cultures were kept for 4 wk under 16/8 h light (~ 130 µE m-2 s-1)/dark at 25±2°C. The regenerated plantlets were used as explants for induction of microtillering.
Multiplication of Embryos
The regenerated shoots of androgenic origin were cultured on MS basal supplemented with different concentrations of BAP, Kn, NAA singly and in various combination (data not presented) to induce and optimize microtillering. Two androgenic plantlets
were inoculated onto each culture tube. In each treatment, 40 androgenic shootlets were cultured.
Culture condition was kept same as used for the regeneration of plantlets from androgenic calli in anther culture experiment.
Encapsulation of Embryos
The in vitro produced embryos, pro-embryos and embryo like structures of androgenic origin were used for encapsulation. Sodium alginate (Sigma Chemical Company, Cat.No. A7003) solution (4% w/v) was prepared by mixing with liquid MS supplemented with 3% sucrose and autoclaved at 121°C under 104 kPa pressure for 15 min. Individual embryos, pro-embryos and embryo like structures were isolated from the clusters under stereomicroscope. Efforts were made to select embryos only. These embryos were mixed with sodium alginate solution for a few seconds, picked up and placed in a sterile aqueous solution of calcium chloride (CaCl2.2H2O; Sigma Chemical Company, USA, Cat.No. 1.02382.0500) with occasional agitation. Calcium alginate beads were formed within 15-20 min. Beads were taken out by decanting off the CaCl2 solution, washed with sterile double distilled water, and surface dried with sterilized blotting paper. Freshly prepared beads were directly cultured on MS fortified with different levels of synthetic hormones (Table 1) and on sterile sand also. Seedling emergence and plantlet regeneration were recorded after 10 d of culture. For each treatment, 40 beads were employed in three replications. The experiment was conducted entirely under control environment using laminar air flow bench.
Results
Callus Induction and Plantlet Regeneration
Cultured anthers of var. 'IR 72' showed profuse callus induction within 15-30 d on N6 supplemented with 2 mg L-1 2,4-D. Most of the responding anthers turned brown and produced 2-4 calli per anther. In a few cases they produced multiple calli. Response of anther to callus formation was 60% and regeneration of green plantlets from calli was only 22.2% (data not presented).
Multiplication of Embryos
Rapid and high rate of multiplication of embryos and pro-embryos (Fig. 1a) were observed on MS containing 2 mg L-l BAP and 4 mg L-l Kn (~ 40.1 embryo and pro-embryo), followed by 4 mg L-l BAP
Fig. 1—Development of synthetic seeds and their germination under in vitro condition: a, stereomicroscopic view of germinating embryos of androgenic origin before emasculation (10×); b, encapsulated embryos in sodium alginate (4%) beads; c, mass germination of beaded embryos on MS medium with no hormone; d, seedling elongation from germinating synthetic seeds; and e, growing plantlet under in vitro culture condition on MS basal medium.
and 1 mg L K n (~ 26.4). BAP singly at a concentra- tion of 4, 6 and 8 mg L-l showed average of 22.6, 26.7 and 14.8 embryos, respectively (data not presented).
Germination of Encapsulated Embryos
Embryos and pro-embryos developed into plantlets on MS basal medium without any phytohormones.
Hence, MS alone was used for artificial encapsulation, followed by germination and plantlet establishment (Figs 1b-e). Beads were cultured on MS supplemented with different concentrations and combination of growth regulators and on sterile sand too (Table 1). The germination varied between 20.0 to 87.5%. Maximum germination (87.5%) was recoded on MS supplemented with 1 mg L-1 BAP, 1 mg L-1 Kn and 0.5 mg L-1 NAA, followed by ½ MS fortified with 2 mg L-1 BAP and 0.5 mg L-1 NAA (62.0%) and full MS with 2 mg L-1 BAP, 0.5 mg L-1 NAA (55.0%).
Minimum germination was observed when the medium was supplemented with 4 mg L-1 Kn (20.0%).
Discussion
High rate of multiplication of embryos, pro- embryos and embryo like structures were recorded on MS supplemented with medium dose (4-6 mg L-1) of BAP. Whereas MS fortified with Kn produced multiple shootlets. This suggests that BAP at high concentration exerts inhibitory effect on shootlet development under in vitro.
The present results indicate that BAP in combination with lower concentrations of NAA increased germination of beaded embryos over control (MS without hormones); whereas, addition of Kn in MS reduced the germination percentage. The germination of unbeaded pro-embryos was 92.5% on MS basal medium. The reduced rate of germination of artificial seeds may be attributed to the damage incurred while separating the embryos from clusters and/or owing to adverse effects of chemicals used for encapsulation. By trail and error, the germination rate may be improvised. The low germination rate of encapsulated embryos of Santalum album15 and Solanum melongena16 was also reported. In contrast, however, superiority of encapsulated embryos was reported in horseradish6, microspore-derived embryos of barley17 and in rice7. The germinated artificial seeds produced roots and established into complete plantlets on transfer to MS basal medium. Moderate germination (40.0%) was observed when the artificial seeds were evaluated for germination on sterile sand in vivo.
Recent advances in the production of artificial seeds revealed that beside somatic embryos, encapsulation of cells and tissues developed in vitro is becoming popular. It offers a simple way of handling cell and tissues, protecting them against strong external gradients and as an efficient delivery system17. Brar et al18 emphasized the need for intensive research on artificial seeds and outlined its impact on mass propagation of true breeding hybrids.
Storage of encapsulated embryos for a considerable period of time allows the preservation of valuable elite germplasm. The judicious and intelligent coupling of artificial seed technology with that of microcomputer would be important in achieving the automated encapsulation and regeneration of plantlets. This would tremendously increase the efficiency of encapsulation and production of homogenous and high quality artificial seeds, and deems to be revolutionizing the current concept of commercial micro-propagation method19. It is important to mention, though, that information on the development of synthetic seeds from anther-derived embryos is scanty. Therefore, the present findings would generate a new vista for rapid multiplication of androgenic embryos and production of uniform synthetic seeds. Encapsulation of embryos makes easier shipment from one laboratory to another and storage of elite materials for research purposes.
Furthermore, synthetic seeds are expected to offer an
Table 1—Germination of synthetic seeds of androgenic origin on different media under in vitro and in vivo conditions Medium and treatment No. of synthetic
seeds germinated*
Germination (%)
½ MS + 2 mg L-1 Kn + 0.5 mg L-1 NAA
21d 52.5
MS + 2 mg L-1 Kn + 0.5 mg L-1 NAA
15f 37.5
½ MS + 2 mg L-1 BAP + 0.5 mg L-1 NAA
26c 62.0
MS + 2 mg L-1 BAP + 0.5 mg L-1 NAA
22d 55.0
½ MS only 19e 47.5
MS + 1 mg L-1 BAP + 1 mg L-1 Kn + 0.5 mg L-1 NAA
35b 87.5
MS + 4 mg L-1 Kn 08g 20.0
Sterile sand 16ef 40.0
Unbeaded embryos on MS basal
37a 92.5
*Values bearing same letter in the column are not significantly different at P=0.05 of DMRT
appropriate recipient system for alien gene transfer in micro-projectile based gene delivery system which of course has to be explored in course of time.
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