Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
  • A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
  • A01H4/002—Culture media for tissue culture

Definitions

• This invention refers to a field of plant biotechnology, specifically to a new process for the induction of direct in vitro organogenesis in onion, with a specific application use in micropropagation or genetic transformation in onion.
• Onion (Allium cepa L.) is the second most important vegetable species worldwide and is produced in almost all climatic regions. It can be multiplied by seeds, sets or vegetatively. Vegetative in vitro methods are used for multiplication of valuable breeding lines including maintaining male sterile lines, used in hybrid seed production. Methods of direct in vitro organogenesis can be further used for successful genetic transformation in onion.
• the claimed invention is a process for induction of direct in vitro organogenesis in onion, comprising the steps of:
• the applied differentiation and induction media contain as solidifiers gellan-gum, mixture of gellan-gum and agar, or only agar.
• the duration of the growth on induction media is from 3 to 12 days.
• the induction and differentiation media contain sucrose, glucose or maltose as a source of carbohydrates.
• the induction and/or differentiation media contain 25-100 g/1 of sucrose.
• the induction media contain 2,4-dichlorophenoxyacetic acid or picloram as a sources of auxins.
• the induction media contain auxin or auxin and 6-benzylaminopurine, thidiazuron or isopentenyladenin (2ip) as sources of cytokinins.
• the differentiation media contain thidiazuron or 6-benzylaminopurine as sources of cytokinins.
• the induction and differentiation steps are performed in light or in darkness.
• composition of macro and micro elements in induction and differentiation media corresponds to BDS media prepared according to Dunstan and Short, 1977, B5 media
• the plant material used in the following Examples originated from different, publicly available sources, cultivars were received from genebanks or were purchased at retail, and inbred lines were received from the US public breeding program (Dr. M.J. Havey, USDA, Madison, Wisconsin, USA).
• Various genotypes of onion were used in these experiments: Belokranjka (Slovenia), Ptujska rdeca (Slovenia), Stuttgarter Riesen, Timor, Shenshu Yellow, Yamaguchi Koudaka, Texas Early Grano 502, experimental hybrid XPH 3371 FI (Asgrow), hybrids 70723 (B1717BxB2923B) and 70719 (B2371CxB2923B), inbred lines B2355B, B2923B, MSU2935B, MSU5718B, MSU8155B.
• flower buds were cultured in Petri dishes 100 mm in diameter (30 per dish) on induction media described later. The flowers were on induction media for 6 days (where not said otherwise). After the induction period, flowers were subcultured on Petri dishes containing differentiation media, as described in the following paragraphs.
• the embodiment 2 differed from embodiment 1 in that the flowers were cultured on induction media and were extracted (perianth removed) before transfer to differentiation media ovaries.
• Petri dishes were sealed with ParafilmTM (American National Can, Greenwich, CT, USA) to prevent evaporation and exposed to a 16/8 hours photoperiod at 21-23 °C and illumination of approximately 80 ⁇ mol m "2 s " '.
• ParafilmTM American National Can, Greenwich, CT, USA
• Such shoots were divided and subcultured on the elongation media, on which they elongated and produced normal plantlets. A proportion of these organogenic structures remained as nodular bumps. When such clusters were subcultured on hormone free media, elongation of shoots occurred, although the organogenic potential was preserved for at least 3 subcultures.
• the most suitable medium for elongation of shoots was basal medium (or half strength basal medium) excluding phytohormones, with the concentration of sucrose or glucose lowered (20-70g/l, recommendable concentration 30g/l).
• Basal medium or half strength basal medium
• concentration of sucrose or glucose lowered (20-70g/l, recommendable concentration 30g/l.
• the media could additionally contain auxins such as 0.5-1.0 mg/1 indolebutyric acid (IBA).
• IBA indolebutyric acid
• the duration of the induction stage had a significant effect on regeneration.
• the highest shoot regeneration was observed on ovaries that were placed on differentiation media after 6 days. Shorter or longer induction treatment resulted in decreased regeneration. At 6 days, the lowest hyperhydration also occurred.
• sucrose concentration in induction and differentiation media was studied using 3 genotypes and embodiment 2.
• Three different concentrations of sucrose used were: 100 g/l (Il/Dl), 50 g/l (I4/D4) and 25 g/l (I5/D5).
• the results are presented in Table 4.
• auxin composition in induction media was studied.
• Induction media contained 2 mg/1 2,4 D (14), 1 mg/1 picloram (18), 2 mg/1 picloram (19) or 5 mg/1 NAA (110).
• the differentiation media were Dl and D4. The results are presented in Table 5.
• Induction media contained 2 mg/l BAP (14), cytokinin omitted (11 1), 1 mg/1 TDZ (112) and 2 mg/1 2ip (113).
• the differentiation medium was D4. The results are presented in Table 6.
• the highest regeneration was obtained on medium 112, the difference was statistically significant compared to 111 and 113 (first genotype), with the second genotype the highest regeneration was obtained on 14 medium.
• the percentage of hyperhydrated shoots was also low on 112 medium. Omitting cytokinin in the induction media (111) greatly reduced regeneration.
• Induction and differentiation media included BDS medium (I4/D4), B5 medium (I14/D12) or MS medium (I15/D13). The results are presented in Table 9. Table 9:

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• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Biotechnology (AREA)
• Developmental Biology & Embryology (AREA)
• Cell Biology (AREA)
• Botany (AREA)
• Environmental Sciences (AREA)
• Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)

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Cited By (5)

Citations (1)

• 1998
  • 1998-09-24 SI SI9800247A patent/SI20053A/sl unknown
• 1999
  • 1999-09-22 AU AU57685/99A patent/AU5768599A/en not_active Abandoned
  • 1999-09-22 WO PCT/SI1999/000022 patent/WO2000016610A1/en not_active Ceased

Patent Citations (1)

Non-Patent Citations (5)

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