Agricultural Research Management Information System

Home
Research Summary
Search
About Us
- ARMIS
- Brochure
Contact Us
Report
User Request
Data Input
Help
- Operation Manual
  - PDF
  - Video
- Program Area & Commodity
We have reached 37600 number of research entries at this moment.

- Logout

Research Detail

Home
Research
Detail

In Vitro Development of Cauliflower Synthetic Seeds and Development of Plantlets in Vivo

Zahida Qamar
National Centre of Excellence in Molecular Biology, University of the Punjab, West Canal Bank Road Thokar Niaz Baig, Lahore 53700, Pakistan

Md. Belal Hossain
Department of Plant Pathology, Sher e Bangla Agricultural University, Dhaka

Idrees A. Nasir
National Centre of Excellence in Molecular Biology, University of the Punjab, West Canal Bank Road Thokar Niaz Baig, Lahore 53700, Pakistan

Bushra Tabassum
National Centre of Excellence in Molecular Biology, University of the Punjab,West Canal Bank Road Thokar Niaz Baig, Lahore 53700, Pakistan

Tayyab Husnain
National Centre of Excellence in Molecular Biology, University of the Punjab, West Canal Bank Road Thokar Niaz Baig, Lahore 53700, Pakistan

Abstract/ Executive Summary:

Synthetic seeds of cauliflower cv. Chillout were developed by encapsulating mature somatic embryos in neutral gel media. Somatic embryos were obtained by optimizing callus and cell suspension cultures of cauliflower. Friable, yellowish embryogenic calli were obtained on MS supplemented with 2 mg/l 2,4?D and 0.5 mg/l BAP using hypocotyl as explants, while calli were regenerated in media consisting of 5 mg/l BAP, 2 mg/l Kn and 6 mg/l GA₃. Somatic embryogenesis was induced in cell suspension culture where auxins were removed in successive steps triggering conversion of globular cells into the heart, torpedo stage (71%) and finally into cotyledonary/somatic embryos (28%). The mature somatic embryos were encapsulated by mixing mature cell suspension with sodium alginate and calcium chloride mixture (1 : 4). Developed synthetic seeds germinated into complete plantlets when placed in neutral gel media. Germination efficiency of synthetic seeds decreased to about 50 per cent after 12 weeks of storage at 4ºC followed by a rapid decrease to zero per cent after 16 weeks. It was also observed that cauliflower plantlets from synthetic seeds survived successfully when transferred to soil demonstrating that cauliflower synthetic seeds is a promising step towards their in vivo direct use.

Keywords: Cauliflower, Hypocotyl, Synthetic seeds, Encapsulation

Location:

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Cauliflower

Objectives:

The major objective of this present study was i) to establish an efficient callus induction protocol from hypocotyl for cell suspension ii) to produce cauliflower synthetic seeds. The assessment of germination viability of synthetic seeds and the establishment of plantlets from synthetic seeds in vivo was another aim of this study

Methodology:

Cauliflower seeds of cv. Chillout were placed on a sterile moist filter paper in Petri plates and incubated in the dark at 25 ± 2°C for germination. Before plating, seeds were surface sterilized with 0.1% mercuric chloride solution for 5 min followed by washing with sterile distilled water three times, 5 min each under aseptic condition. The hypocotyl explants from 5 to 7 days old seedlings were used for callus formation. The hypocotyls were cut into 0.3 to 0.6 cm long pieces and placed horizontally in callus induction MS supplemented with 2 mg/l 2,4 D, 0.5 mg/l BAP, 3% sucrose and solidified with phytagel; the pH of the medium was adjusted at 5.7 to 5.8 before autoclaving. Proembryogenic callus was obtained approximately after 15 days with regular sub-culturing at 7 days interval. Initially the cultures were maintained in the dark for 3 to 5 days and subsequently placed under 16/8 hrs photoperiod. To check the shoot regeneration response of cauliflower, the established embryogenic calli were placed in various shoot regeneration media, each supplemented with different hormonal supplements. The best combination of regeneration media was 4 to 6 mg/l BAP, 2 ? 4 mg/l Kn and 4 to 6 mg/l GA3 fortified with MS vitamins (30 μM adenine sulphate, 3 μM thiamine HCl and 580 μM NaH₂PO₄). The cell suspension of the embryogenic callus was established in the callus medium initially for one week, followed by subculturing it in the same medium without 2,4 D. For this purpose, 250 ? 300 mg of calli were placed in 30 ml of cell suspension media and placed on a rotary shaker at 90 rpm under 16 hrs photoperiod at 22 ± 2°C. Cells were regularly observed under the microscope (Olympus U?PMTVC 3B15937, Japan). For synchronization to achieve uniform cell suspension and to remove dead cell clumps, cells were sieved through a stainless sieve of 150 μm pore size mesh. Synthetic seeds were stored at 4ºC. Their germination viability was checked after 16 weeks of their storage. For germination assessment, neutral gel consisting phytagel dissolved in autoclaved tap water was used. Complete plantlets regenerated from synthetic seeds were transferred into pot containing autoclaved compost soil under controlled conditions. The compost soil was prepared by thoroughly mixing clay, sand and compost in the ratio of 1 : 1 : 1 respectively. The plants were completely covered with plastic bag for 2 weeks to maintain the humidity and then progressively exposed to normal environmental conditions for acclimation

Source of Information: Plant Tissue Cult. & Biotech. 24(1): 27-36, 2014 (June)

Article Link (Online Journal):

Funding Source:

1.

Budget:

Total Budget (Tk.) :

Achievement/Findings:

After regeneration in a controlled environment, the developing plantlets were transferred into pots containing autoclaved compost soil. The optimal conversion rate and viability were obtained in 1 : 1 : 1 of clay, sand and compost, respectively. In several earlier studies researchers investigated the possibility of sowing synthetic/artificial seeds in soil, for example, using vermiculite, sand and soil for M.26 apple rootstock and Citrus reticulata and the use of sand for elite indica rice. It needs to be emphasized that the optimal conditions need to be determined empirically for each species examined. This is the first successful study of cauliflower synthetic seed production from the hypocotyl, successful germination and regeneration of synthetic seeds into complete plantlets. The main objective of the present study was to optimize an inexpensive and yet a sophisticated method for synthetic seed production of cauliflower with a prolonged period of viability capable of germinating and developing into viable plants. Synthetic seed technology seems to be a promising method in plant tissue culture industry. This method is very advantageous, especially for those plant species which do not produce seeds or for a long term storage of elite genotypes selected through traditional breeding methods or genetically engineered plants.

Knowledge Management: Journal