Efficient protocols for in vitro regeneration of Pennisetum glaucum (L) Br

A system was developed for in vitro regeneration of Pennisetum glaucum through organogenesis and somatic embryogenesis. Mature embryo and leaf base explants of Pennisetum glaucum (L) Br. cv HH B60 (Poaceae) were cultured on Murashige and Skoog agar medium supplemented with 11.3 microM of 2,4-D for callus induction. Embryogenic calli were induced within eight weeks. Percentage of callus induction and somatic embryogenesis was significantly higher in mature embryo than leaf base explants. Maximum shoot regeneration was obtained via organogenesis on MS medium supplemented with 4.43 microM of BAP and 4.64 microM of kinetin from the calli of both the explants. The frequency of plant regeneration through somatic embryogenesis was comparatively lower than organogenesis. Regeneration frequency was higher in mature embryo explants than leaf base explants. The shoots regenerated via organogenesis were elongated and rooted efficiently on MS medium supplemented with IBA (0.49 microM). The rooted plantlets were hardened and transferred to soil.     

Somatic embryogenesis and plant regeneration of Gladiolus (Gladiolus hort.)

Summary Friable embryogenic callus and somatic embryos of 4 Gladiolus cultivars were obtained on Murashige and Skoog (MS) medium with various concentration of auxins from the following explants: corm slices, young leaf bases and whole, intact plantlets. Somatic embryos transferred on MS hormone-free medium regenerated into plantlets. All plantlets obtained through embryogenesis did not differ phenotypically from the parental clones. The embryogenic friable callus has been maintained for over 2 years in culture and has retained a very high regeneration capacity.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - KIN kinetin - NAA naphthaleneacetic acid - MS Murashige and Skoog Medium (1962) - E embryogenic callus - NE non-embryogenic callus     

Plant regeneration via somatic embryogenesis in chickpea (Cicer arietinum L.)

Efficient plant regeneration via somatic embryogenesis has been developed in chickpea cultivar C235. Leaf explants, on MS medium supplemented with 1.25 mg/l 2,4-D and 0.25 mg/l kinetin, yielded somatic embryos with high efficiency during dark incubation. MS medium supplemented with B₅ vitamins, 0.125 mg/l IBA and 2 mg/l BAP was found suitable for embryo maturation. The well formed embryos germinated into plantlets on basal B₅ medium supplemented with 0.25 mg/l BAP. Further development into healthy plantlets was obtained on basal B₅ medium. Hardened plantlets produced normal, fertile plants upon transfer to soil.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-Benzyl-aminopurine - IAA IndoIe-3-acetic acid - IBA Indole-3-butyric acid - NAA 1-Naphthalene acetic acid - Kinetin 6-furfuryl aminopurine - Zeatin 6-(4-hydroxy-3-methylbut-2-enylamino)-purine     

De novo shoot regeneration from root cultures of Garcinia indica Choiss

Roots of plantlets of Garcinia indica when cultured for long time on half strength MS medium supplemented with BAP (0.44-2.22 microM) showed production of de novo shoots. Roots attached to mother plant showed more number of shoots, while excised root segments produced lesser shoots. Shoots (0.5-0.8 cm) were transferred to elongation medium consisting of Woody Plant Medium (WPM) supplemented with BAP (4.44-22.69 microM), IAA (5.71 microM) and kinetin (4.65 microM). It was observed that shoot length increased to 1-2 cm. WPM medium supplemented with NAA (2.69-10.74 microM) and IBA (4.90 microM) induced rooting within 20-25 days. Using the present protocol, 20-25 plantlets could be regenerated from single root explant within 3 to 4 months. The protocol has potential for large scale production of elite plants.     

High Frequency Somatic Embryogenesis in Cotton

A highly reproducible system for efficient somatic embryogenesis was developed to regenerate plantlets from cotton (Gossypium hirsutum L.) cultivars (Nazilli M-503 and Nazilli 143). Shoot apices, hypocotyls and nodes of 10-d-old seedlings were used as explants. High frequency (100 %) embryogenic calli was initiated from all tested explants on Murashige and Skoog (1962) (MS) media supplemented with 1 g dm^–3 polyvinylpyrrolidone (PVP), 1 mg dm^–3 6-benzylaminopurine (BAP), 0.5 mg dm^–3 kinetin for Nazilli M-503 and 1 g dm^–3 PVP, 2 mg dm^–3 BAP, 0.5 mg dm^–3 kinetin for Nazilli-143. Globular stage somatic embryos were produced 4 months after transfer to hormone-free MS medium supplemented with 1 g dm^–3 PVP. Subsequent subculture of globular embryos every 3 weeks on hormone-free MS medium led to the development of torpedo and cotyledonary stage embryos with the frequency of 75 and 83.2 % from hypocotyl explants of Nazilli M-503 and Nazilli-143, respectively. Afterwards, mature somatic embryos were isolated and cultured on hormone-free MS medium for germination and development into plantlets. The highest germination frequency (42.9 %) for Nazilli M-503 somatic embryos were obtained on hormone-free MS medium after 5 months with hypocotyl explants, whereas germination frequencies of Nazilli-143 embryos from hypocotyl, node and apex explants varied between 22 – 30 %.     

Callus culture of Coronilla varia L. (crownvetch): plant regeneration through somatic embryogenesis

Callus culture and plant regeneration through somatic embryogenesis have been obtained in Coronilla varia. Media used were UM (25) supplemented with 2 mg/l 2,4-D followed by subculture on MS (18) containing 1 mg/l 2-iP and 0.1 mg/l IAA. Embryoids developed into complete plantlets on filter paper saturated with hormone-free MS medium.     

High frequency plant regeneration of Cymbopogon martinii (Roxb.) Wats by somatic embryogenesis and organogenesis

Embryogenic callus cultures were obtained upon repeated sub-culture of non-embryogenic callus from nodal segments of Cymbopogon martinii (Roxb.) Wats. Murashige and Skoog's medium supplemented with 1mg/l 2,4-dichlorophenoxyacetic acid and 0.5 mg/l kinetin and Linsmaier and Skoog's medium supplemented with 2mg/l 2,4-dichlorophenoxyacetic acid and 0.4 mg/l kinetin were used as maintenance media for non-embryogenic and embryogenic cultures, respectively. Plant regeneration occurred through organogenesis in MS basal media containing 2 mg/l kinetin, 1 mg/l 6-benzylaminopurine, 0.2 mg/l biotin, 0.2 mg/l Ca-pantothonate and 0.1 mg/l napthalene acetic acid. Embryogenesis was induced in LS medium supplemented with 1 mg/l kinetin, 0.5 mg/l 6-benzylaminopurine and 0.1 mg/l 3-indole acetic acid. Plant regeneration at high frequency was recorded both through organogenesis and embryogenesis in different passages of long term callus cultures.Abbreviation MS Murashige and Skoog medium - LS Linsmair and Skoog medium - BAP 6-benzylaminopurine - kin kinetin - 2,4-D 2,4-Dichlorophenoxyacetic acid - IAA Indole-3-acetic acid - CH Casein hydrolysate - CaP calcium pantothonate - NAA napthalene acetic acid     

Production of haploids of neem (<Emphasis Type="Italic">Azadirachta indica</Emphasis> A. Juss.) by anther culture

Androgenic haploids of the neem tree (Azadirachta indica A. Juss.) were produced by anther culture at the early- to late-uninucleate stage of pollen. Haploid formation occurred via callusing. The best medium for inducing callusing in the anther cultures was Murashige and Skoog's basal medium (MS) (9% sucrose) supplemented with 1 microM 2,4-D, 1 microM NAA and 5 microM BAP, while anther callus multiplied best on MS medium supplemented with 1 microM 2,4-D and 10 microM Kn. These calli differentiated shoots when transferred to a medium containing BAP; 5 microM BAP was optimum for young calli (75% cultures differentiated shoots), but older calli showed the best regeneration with 7.5 microM BAP. Shoots elongated at a lower concentration of BAP-0.5 microM. These shoots were multiplied by forced axillary branching and rooted in vitro. The plants were subsequently established in soil. Of the plants that regenerated from anther callus 60% were haploid, 20% were diploid and 20% were aneuploid.     

Plant regeneration of Sapindus trifoliatus L. (soapnut) through somatic embryogenesis

Somatic embryogenesis and subsequent formation of plantlets was obtained from callus cultures derived from leaves of mature (over 60years old) Soapnut (Sapindus trifoliatus L.) tree. Callus was induced from leaf explants and grown on Murashige and Skoog's medium supplemented with 2,4-dichlorophenoxy acetic acid (2,4-D) and kinetin. Reduction of 2,4-D concentration during subsequent subcultures resulted in formation of embryoids. These embryoids developed further when transferred to a medium containing benzylaminopurine and kinetin and then to a hormone-free medium. Unless 5-methyl tryptophan was added and the level of sucrose raised, the embryoids began to recallus and failed to form plantlets.     

Stimulatory Effect of Partial Desiccation on Plant Regeneration in Indica Rice (Oryza sativa L)

A simple in vitro protocol was established for high frequency plant regeneration via organogenesis and somatic embryogenesis from the callus cultures derived from immature inflorescence segments of indica rice (Oryza sativa L cvs Safari-17 and Kasturi). Embryogenic and nodular calli were initiated on MSB medium supplemented with 2, 4-D and sucrose (3.0%, w/v). Somatic embryogenesis occurred after transfer of embryogenic calli to MSB medium containing 2.25 μM 2,4-D, 2.2 μM BAP, 2.9 μM thiamine HCl and 244.86 μM L-tryptophan. Plantlet/shoot regeneration occurred after transfer of embryogenic calli to MSB medium containing 17.6 μM BAP and 1.12 μM 2,4-D. Partial desiccation (up to 12, 24, 48, 72 and 96 h) of embryogenic calli prior to transfer to regeneration medium stimulated regeneration frequency. Highly significant (P<0.001) difference was observed for regeneration frequency and average number of plantlets/shoots regenerated per callus in partially desiccated calli in comparison to non-dehydrated calli. Regeneration frequency increased from 33.3% to 80% after 24 h of desiccation treatment to callus cultures of cv. Safari-17, and from 46.7% to 93.3% after 48 h of desiccation treatment to callus tissues of cv. Kasturi. Regenerated shoots were rooted on MSB medium supplemented with 4.9 μM IBA. Plants with well-developed roots were transferred to pots where they grew well and attained maturity.     

An efficient and reproducible method for regeneration of whole plants from mature seeds of a high yielding Indica rice (Oryza sativa L.) variety PAU 201

Tissue culture is one of the tools necessary for genetic engineering and many other breeding programs. Moreover, selection of high regenerating rice varieties is a pre-requisite for success in rice biotechnology. In this report we established a reproducible plant regeneration system through somatic embryogenesis. The explants used for regeneration were embryogenic calli derived from mature seeds cultured on callus induction media. For callus induction mature seeds were cultured on MS medium containing 30 g/l sucrose combined with 560 mg/l proline and 1.5-3.5 mg/l 2,4-D and 0.5-1.5 mg/l Kin. For plant regeneration, embryogenic calli were transferred to MS medium containing 30 g/l sucrose, supplemented with 1.0-3.0 mg/l BAP, 0.5-1.5 mg/l Kin and 0.5-1.5 mg/l NAA. The highest frequency of callus induction (44.4%) was observed on the MS medium supplemented with 2.5 mg/l 2,4-D, 0.5 mg/l Kin, 560 mg/l proline and 30 g/l sucrose. The highest frequency of shoot regeneration (42.5%) was observed on the MS medium supplemented with 2.0 mg/l BAP, 0.5 mg/l NAA and 0.5 mg/l Kin. The plantlets were hardened and transferred to soil in earthen pots. The developed method was highly reproducible. The in vitro developed plants showed normal growth and flowering under glasshouse conditions.     

Effect of genotype, gelling agent, and auxin on the induction of somatic embryogenesis in sweet potato (Ipomoea batatas Lam.)

Lateral buds of six cultivars of sweet potato were induced to form embryogenic callus in a culture medium solidified with two types of gelling agents, Agar or Gelrite, and supplemented with various concentrations of auxins, 2,4-D, 2,4,5-T and Picloram. Of the six cultivars screened, only three gave an embryogenic response. Best results with an average of 3.53% embryogenic response were obtained with the medium solidified with Agar, while in Gelrite only 0.45% of lateral buds gave rise to embryogenic callus. The interaction between the genotype and auxins was highly significant; particularly the optimal response was obtained with cv. Zho and 865 yielding 10.7 and 14.7% somatic embryogenesis, respectively, in the medium containing 2,4,5-T or Picloram. The plant conversion was dramatically improved by subculture of the embryogenic callus on the medium with the combination of 1 microM 2,4-D and 1 microM Kinetin or 5 microM ABA alone before transfer of mature embryos onto hormone-free medium. The embryogenic callus of sweet potato and its sustained ability to further regenerate plants have regularly been maintained for several years by frequent subculture in 5 microM 2,4,5-T or the combination of 10 microM 2,4-D and 1 microM BAP or kinetin. The embryo-derived plants seemed apparently genetically stable and similar to the hexaploid parental plants, based on morphological analysis and their ploidy level determined by using flow cytometry.     

Somatic embryogenesis from immature and mature embryos of a minor millet Paspalum scrobiculatum L.

Immature and mature zygotic embryos of Paspalum scrobiculatum L. cv. PSC 1 cultured on MS or N₆ nutrient medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), formed embryogenic callus. Induction of embryogenic callus and subsequent somatic embryogenesis was possible at a lower concentration of 2,4-D on N₆ than MS medium. Immature embryos were highly totipotent, forming somatic embryos at a higher frequency than mature embryos. Addition of amino acids (L-proline or L-tryptophan) to 2,4-D medium resulted in significant enhancement of embryogenesis on culture of mature embryos. Silver nitrate also supported an increased frequency of embryogenesis. Thus it is possible to have high frequency of somatic embryogenesis on culture of mature embryos, which are available in abundance and with ease than immature embryos. The somatic embryos readily germinated and formed plantlets on hormone-free regeneration medium. The regenerated plantlets were successful on transfer to soil and set seed.     

Somatic embryogenesis and plant regeneration from leaf callus of Ocimum basilicum L

A effective protocol for complete plant regeneration via somatic embryogenesis has been developed for Ocimum basilicum L. Callus was initiated from leaf explant of young plant on supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) 1.0 mg l(-1), 3% sucrose and 0.9% agar. The calli showed differentiation of globular structure embryos when transferred to MS medium containing 2,4-D 0.5 mg l(-1) and BAP 1.0 mg l(-1). The maximum globular structure embryos were further enlarged and produced somatic embryos in MS basal medium supplemented with BAP 1.0 mg l(-1)+NAA 1.0 mg l(-1) + KN 0.5 mg l(-1). Continued formation of globular embryo and germination of embryos occurred in this medium. Complete plantlets were transferred onto specially made plastic cup containing soilrite followed by their transfer to the garden soil. Survival rate of the plantlets under ex vitro condition was 80%.     

Effect of 2,4-d, hydric stress and light on indica rice (Oryza sativa) somatic embryogenesis

With the purpose of increasing the embryogenesis regeneration process in vitroplants obtained from somatic embryos of the indica rice variety CR-5272 (Oryza sativa L.), two independent experiments were performed. The first experiment consisted in the effect of combination of three concentrations of the gelling agent Phytagel (1.8. 2.4. and 3 gL(-1)) and four 2,4-D concentrations (2.26, 4.52. 6.78, and 9.05 microM) on the induction and subsequent regeneration of embryogenic calli. On the second experiment, the pre-regeneration phase was modified: calli were subjected to darkness or diffuse light conditions for one, two, and three weeks. In embryogenesis induction, 35% calligenesis was obtained using the MS culture medium supplemented with 6.78 microM of 2.4-D and 2.4 gL(-1) Phytagel, whereas on the control treatment (MS medium supplemented with 9.05 microM of 2.4-D and 3 gL(-1) Phytagel ) 24% calligenesis was obtained. In addition, regeneration percentages were improved (22% and 16% for calli induced with the above treatments, respectively). Furthermore, in light exposure experiments, the best result was obtained by exposing the embryogenic calli to darkness for one week in pre-regeneration, followed by direct light exposure during the regeneration phase.     

Somatic embryogenesis from mesocotyl and leaf-base segments of <Emphasis Type="Italic">Paspalum scrobiculatum</Emphasis> L., a minor millet

Summary Somatic embryos could be induced from embryogenic callus originating from mesocotyl as well as leaf-base segments of Paspalum scrobiculatum on Murashige and Skoog (MS) or Chu et al. (N₆) medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D; 4.5, 9.0, 18.0, and 22.5 μM). N₆ medium was better than MS, for both explants, for high-frequency somatic embryogenesis. Also, mesocotyl tissues were relatively more totipotent than leaf-base segments. The somatic embryos ‘germinated’ and formed plantlets on transfer of embryogenic calluses to hormone-free MS or N₆ regeneration medium. Embryogenic cultures could be maintained on low hormone medium which readily regenerated to form plantlets on hormone-free medium. A higher frequency of plantlet formation occurred on MS than on N₆ medium. In vitro-formed plantlets were gradually acclimatized in the culture room and on transfer to soil flowered and set seed. Somatic embryogenesis and plantlet regeneration from mesocotyl and leaf-base segments are potentially simpler systems than regeneration from ‘embryonic’ explants such as immature embryos and unemerged inflorescences.     

Totipotency of coleoptile tissue in indica rice (Oryza sativa L. cv. ch 1039)

Embryogenic and non-embryogenic calluses were induced from 3,4,5 and 7d old coleoptile segments of indica rice (Oryza sativa L. cv. CH 1039). Compact, globular, yellow and creamy embryogenic and white friable non-embryogenic callus arose from the cut end and entire length of the coleoptile segments. Murashige and Skoog's (MS) medium supplemented with 2.5mg/1 2,4-D was used as callus induction medium. Plant regeneration from coleoptile segments occurred with the transfer of embryogenic callus to MS basal medium supplemented with 2.0mg/1 BAP and 0.5mg/1 NAA in combination. Average number of regenerated plants from one coleoptile ranged from9.1 to 14.0.Four day old coleoptiles showed the highest frequency of plant regeneration.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - MS Murashige and Skoog (1962) - NAA 1-naphthalene acetic acid     

Rapid multiplication of Polyscias filicifolia by secondary somatic embryogenesis

Efficient plant regeneration through somatic embryogenesis was achieved in Polyscias filicifolia. Embryogenic calluses were induced on Murashige and Skoog (MS) basal medium supplemented with 0.5 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg l⁻¹ benzylaminopurine (BAP; type I callus) and on MS medium with 2.0 mg l⁻¹ 2,4-D and 0.01 mg l⁻¹ kinetin (type II callus) from leaf explants of a 2-yr-old plant. Primary somatic embryos (PSEs) developed after four passages of suspension culture established from embryogenic callus when cultured in liquid half-strength MS medium (1/2 MS) without growth regulators. PSEs in the cotyledonary stage were multiplied by adventitious embryogenesis. Single secondary somatic embryos (SSEs) or their clusters developed at the base of PSE hypocotyls and regenerated into plantlets in a one-step process on plant growth regulator-free 1/2 MS medium. Low sucrose concentration of 15 g l⁻¹ promoted development of normal SSEs. All SSEs regenerated into single, well-rooted plantlets on a Nitsch and Nitsch medium supplemented with 0.5 mg l⁻¹ kinetin, 0.1 mg l⁻¹ indole-3-butyric acid, and 10 mg l⁻¹ adenine sulfate. Subsequent two subculture cycles on the same medium were necessary to obtain plantlets sufficiency developed to allow successful transfer to the soil. Rooted plantlets were established in a peat mixture with 90% survival, with the plants showing normal morphological characteristics.     

Direct somatic embryogenesis and plant regeneration from mature sugarbeet (Beta vulgaris L.) zygotic cotyledons

An in vitro protocol has been developed for direct somatic embryogenesis of zygotic cotyledons from mature sugarbeet (Beta vulgaris L.) embryos. Explants were sequentially cultured on modified Murashige and Skoog (MS) medium supplemented with different combinations of 2,4-D, NAA, BAP and TIBA. Somatic embryogenesis was induced within 4 weeks of culture on embryogenesis induction medium which contained MS medium supplemented with BAP and TIBA. Proliferation of somatic embryos was observed on embryo proliferation medium, which contained MS medium supplemented with BAP and NAA within 4 weeks of culture. Plants were regenerated on hormone free half; strength MS medium containing a low sucrose concentration. With some sugarbeet lines, high frequencies of plant regeneration in excess of 90percnt; were observed. The incorporation of TIBA in the media was essential for successful regeneration.     

Direct somatic embryogenesis and plantlet regeneration from leaf explants of niger, Guizotia abyssinica (L.f.) Cass

Somatic embryogenesis was induced in niger (Guizotia abyssinica (L.f.) Cass.) using a simple one step method. Leaf explants were cultured on MS medium with 2,4-D and BAP/Kn individually and in combination. Somatic embryogenesis occurred directly without an intervening callus proliferation phase from subepidermal regions of leaf explants on MS medium supplemented with 2,4-D (2.0 and 5.0 microM) plus Kn/BAP (1.0 and 2.0 microM). Regenerated somatic embryos were successfully grown into whole plants.