Regeneration via somatic embryogenesis and organogenesis in different cultivars of cotton (Gossypium SPP.)

Summary In vitro studies related to somatic embryogenesis and organogenesis were performed in different cultivars of cotton. Gossypium hirsutum cultivars SH-131, LH-900, Hybrid H8, Khandwa-2, and Gossypium arboreum cultivars BD-1, BD-6, Sarvottam, and Jawahar Tapti were screened for their ability to regenerate in vitro. Cotyledonary leaves and hypocotyls were used as explants. Vigorous callusing was observed in G. arboreum cultivars. Globular somatic embryos were formed in BD-1, BD-6, Sarvottam, Jawahar Tapti, SH-131, and LH-900. Heart-shaped and torpedo stages were also observed. Embryos of BD-1 and BD-6 germinated and formed distincts shoot and root poles. 2-Isopentenyladenine (2iP) was effective in the induction of somatic embryos. Hybrid H8 and Khandwa-2 regenerated by directly forming shoots when cultured on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine and formed roots on hormone-free MS medium     

High frequency regeneration via direct somatic embryogenesis and efficient Agrobacterium- mediated genetic transformation of tobacco

A direct somatic embryogenesis protocol was developed for four cultivars of Nicotiana species, by using leaf disc as an explant. Direct somatic embryogenesis of Nicotiana by using BAP and IAA has not been investigated so far. This method does not require formation of callus tissues which leads to somaclonal variations. The frequency of somatic embryogenesis was strongly influenced by the plant growth hormones. The somatic embryos developing directly from explant tissue were noticed after 6 d of culture. Somatic embryogenesis of a high frequency (87–96%) was observed in cultures of the all four genotypes (Nicotiana tabacum, N. benthamiyana, N. xanthi, N. t cv petihavana). The results showed that the best medium for direct somatic embryogenesis was MS supplemented with 2.5 mg/l, 0.2 mg/l IAA and 2% sucrose. Subculture of somatic embryos onto hormone free MS medium resulted in their conversion into plants for all genotypes. About 95% of the regenerated somatic embryos germinated into complete plantlets. The plants showed morphological and growth characteristics similar to those of seed-derived plants. Explants were transformed using Agrobacterium tumifacious LBA4404 plasmid pCAMBIA1301 harboring the GUS gene. The regenerated transgenic plants were confirmed by PCR analysis and histochemical GUS assay. The transformation efficiency obtained by using the Agrobacterium- mediated transformation was more than 95%. This method takes 6 wk to accomplish complete transgenic plants through direct somatic embryogenesis. The transgenic plantlets were acclimatized successfully with 98% survival in greenhouse and they showed normal morphological characteristics and were fertile. The regeneration and transformation method described herein is very simple, highly efficient and fast for the introduction of any foreign gene directly in tobacco through direct somatic embryogenesis.     

Somatic embryogenesis in suspension cultures of Gossypium klotzschianum anderss

Somatic embryoids differentiated in suspension cultures of G. klotzschianum after 3–4 weeks of culture in a liquid medium containing glutamine (optimally, 10–15 mM). Embryogenesis occurred after a preculture of callus on a medium containing 10 mg/l of the cytokinin, 2iP. The embryoids had meristematic regions, a well formed epidermis, and formed roots and vestigial leaves. Asparagine was much less effective than glutamine in promoting embryoid differentiation. The presence of 2,4-D in the medium resulted in increased vigor of the suspension cultures and subsequently in the formation of many embryoids, but does not seem to be necessary for somatic embryogenesis in cotton.Technical Article 14646 from the Texas Agricultural Experiment Station     

Factors affecting somatic embryogenesis and plant regeneration from a range of recalcitrant genotypes of Chinese cottons (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.)

Summary A new protocol has been developed for the highly efficient somatic embryogenesis and plant regeneration of 10 recalcitrant Chinese cotton cultivars. Calluses and embryogenic calluses were induced on MSB1 medium containing the optimal combination of indolebutyric acid (IBA; 2.46 μM) and kinetin (KT; 2.32 μM). Up to 86.7% of embryogenic calluses differentiated into globular somatic embryos 2 mo. after culture on MSB2 medium containing double KNO₃ and free of growth regultors. Up to 38.3% of the somatic embryos were converted into complete plants in 8 wk on MSB3 medium with l-asparagine (Asn)/l-glutamine (Gln) (7.6/13.6 mM). The plants were successfully transferred to soil and grew to maturity. With the protocol described here, we have obtained hundreds of regenerating plantlets from 10 recalcitrant cultivars, which is important for the application of tissue culture to cotton breeding and biotechnology.     

Plant regeneration via somatic embryogenesis from in vitro tissue culture in two Tunisian Cucumis melo cultivars Maazoun and Beji

Hypocotyl, cotyledon and zygotic embryo explants from two Tunisian Cucumis melo L. cultivars Beji and Maazoun, cultured on the MS medium added with 2,4-D (0.25–1 mg l⁻¹) and BA (0.10–0.50 mg l⁻¹), produce calluses with somatic embryos after 3 weeks of culture. For Beji c.v. the highest percentage (62.50%) of embryogenesis was observed for cotyledons. The average embryo number per callus was 10.40. Embryogenesis induction for zygotic embryos reached 33.50% with 29 embryos per callus. The embryogenesis ability of hypocotyls did not exceed 12.50% (2.50 embryos per callus). Somatic embryogenesis for Maazoun c.v. explants was less efficient. Embryos formation was observed only for cotyledons (29%) and zygotic embryos (25%). Cotyledonary staged embryos, when transferred to hormone free MS medium, germinated. The maximum germination rates were 51.50 and 44.50%, respectively for Maazoun and Beji c.v. The highest percentage (36.50%) of survival plants was noted for Beji c.v. Regenerants were diploids (2n = 2x = 24) and morphologically similar to their parents issued from seeds.     

Evaluation of NemX,a New Cultivar of Cotton with High Resistance to Meloidogyne incognita

The level of resistance to root-knot nematode, Meloidogyne incognita, in NemX, a new cultivar of the Acala-type upland cotton, was evaluated in relation to four resistant breeding lines (N6072, N8577, N901, and N903) and four susceptible cultivars (Maxxa, SJ2, Royale, and Prema). In growth pouch tests, an average of only 4 nematode egg masses was produced on roots of NemX or the resistant lines, compared to a significantly higher average of 21 on the susceptible cultivars. In pot tests, the nematode reproduction factor (RF = Pf/Pi) in NemX and the resistant lines averaged 0.7, compared to a significantly higher average of 10 on the susceptible cultivars. Root galling in NemX or other resistant cotton averaged 15%, compared to 74% on the susceptible cultivars, in either pot or field tests. In plots with low levels of nematode infestation (Pi ≤ 150 second-stage juveniles [J2]/500 g soil), lint yield of NemX averaged 1,370 kg/ha and was less than the yield of susceptible Maxxa (1,450 k g /h a ). However, in plots with medium or high levels of nematode infestation (Pi = 151-300 or >300 J2/500 g soil, respectively), yields of NemX decreased only slightly and averaged 1,300 or 1,050 kg/ha, respectively, whereas yields of Maxxa were severely reduced to 590 or 503 kg/ha, respectively. Fusarium wih symptoms were observed on both NemX and Maxxa, and percent occurrence increased with increasing preplant nematode density. In plots with the highest nematode densities, 22% of NemX and 65% of Maxxa plants were wilted. NemX was highly effective against five M. incognita isolates and moderately effective against a sixth isolate that had been exposed to resistant cotton over several seasons. These results showed that NemX is as resistant to M. incognita as the four breeding lines, and much more resistant than the tested susceptible cultivars of cotton.     

Induction of somatic embryogenesis in gum arabic tree [Acacia senegal (L.) Willd.]

Factors affecting somatic embryogenesis from immature cotyledon of gum arabic tree [Acacia senegal (L.) Willd.] were investigated. Induction of somatic embryogenesis was influenced by plant growth regulator concentrations and addition of amino acids in medium. Best induction of somatic embryogenesis was obtained on MS medium supplemented with 0.45 μM 2, 4-D, 2.32 μM Kin and 15 mM L-glutamine. L-glutamine plays a significant role in the maturation of somatic embryos and most of embryos attained maturity only on L-glutamine (15 mM) containing medium. Maximum percent (75.0 ± 2.5) germination of somatic embryos was recorded on medium containing 0.22 μM BAP.     

TDZ-induced direct shoot organogenesis and somatic embryogenesis on cotyledonary node explants of lentil (Lens culinaris Medik.)

An efficient and simple procedure for inducing high frequency direct shoot organogenesis and somatic embryogenesis in lentil from cotyledonary node explants (without both the cotyledons) in response to TDZ alone is reported. TDZ at concentration lower than 2.0 μM induced shoot organogenesis whereas at higher concentration (2.5–15 μM) it caused a shift in regeneration from shoot organogenesis to somatic embryogenesis. The cotyledonary node and seedling cultures developed only shoots even at high concentrations of BAP and TDZ, respectively. TDZ at 0.5 and 5.0 μM was found to be optimal for inducing an average of 4–5 shoots per cotyledonary node in 93 % of the cultures and 55 somatic embryos in 68 % of the cultures, respectively. The somatic embryos were germinated when transferred to lower TDZ concentration (0.5–1.0 μM). The shoots were rooted on MS basal medium containing 2.5 μM IBA. The plantlets were obtained within 8 weeks from initiation of culture and were morphologically similar to seed-raised plants. The possible role of stress in thidiazuron induced somatic embryogenesis is discussed.Key words: Thidiazuron, Lens culinaris, Somatic embryogenesis, Organogenesis     

In vitro regeneration of Eucalyptus camaldulensis

An efficient in vitro regeneration protocol enables mass multiplication, genetic modification and germplasm conservation of desired plants. In vitro plant regeneration was achieved from nodal segments of 18-months-old superior genotypes of Eucalyptus camaldulensis trees through direct organogenesis (DO) and direct somatic embryogenesis (DSE) pathways. Initial bud break (BB) stage occurred via DO while shoot multiplication phase followed both DO and DSE pathways. Interestingly, both BB and shoot multiplication stages were achieved on shoot induction and multiplication (SIM) media composed of Murashige and Skoog (MS) basal medium supplemented with 2 mg l⁻¹ benzyl aminopurine (BAP) and 0.1 mg l⁻¹ naphthalene acetic acid (NAA). Best shoot elongation response was observed on half strength MS fortified with 0.5 mg l⁻¹ BAP, while root induction and elongation was superior in 1/2 MS + 1 mg l⁻¹ Indole butyric acid (IBA). Full strength MS fortified with cytokinins (BAP) and weak auxin (NAA) in the ratio of 20:1 favored direct regeneration pathways. Further, half strength MS supported shoot and root development. The absence of intervening callus phase in this protocol can help in minimizing the chance occurrence of somaclones. When compared to other compositions tried, hardening in 100 % coco peat resulted in maximum survival (80 %) of the in vitro raised plantlets. For mass multiplication, fortnight subculturing of a single nodal explants for eight passages on SIM medium resulted in 60–148 shoot initials. Repeated subculturing in SIM medium induced the formation of direct somatic embryos which in turn improved the turnover capacity and enabled large scale clonal multiplication of elite and desirable trees of E. camaldulensis. Following this protocol, it takes a minimum time period of four-months between in vitro explant inoculation to hardening stage. In the present study, DO and DSE pathway of plant regeneration was reported occurring simultaneously in the same nodal explants of E. camaldulensis.     

Microspore embryogenesis in anther cultures of two Indian cultivars of Brassica juncea (L.) Czern.

Direct microspore-derived embryo formation in anther cultures of two cultivars of Brassica juncea was obtained. Preliminary culture of anthers at 35°C for 1–5 days prior to maintenance at 25°C stimulated embryogenesis. Embryogenesis was also stimulated by an initial culture at 5°C for 3 days. Analysis of squashed anthers revealed that approximately 10% of the microspores began dividing, but less than 1% developed into macroscopic embryos. All embryos transferred to embryo culture medium survived, but only 30% of these developed directly into normal plantlets. The androgenic plants were haploid (2n=18).     

Evaluation of haemoglobin (erythrogen): for improved somatic embryogenesis and plant regeneration in cotton (Gossypium hirsutum L. cv. SVPR 2)

Somatic embryogenesis in cotton (Gossypium hirsutum L.) is accelerated when the plant regeneration medium is supplemented with haemoglobin (erythrogen). In cotton SVPR 2 lines, a higher frequency of embryoid formation was observed when the medium contained 400 mg/l haemoglobin. Fresh weight of the callus, rate of embryoid induction, number of embryoids formed and the percentage of plant regeneration from somatic embryos were increased. Among the two different cultivars tested, MCU 11 showed no response to the presence of haemoglobin when compared to SVPR 2, and embryogenic callus formation was completely absent in the former. Medium containing MS salts, 100 mg/l myo-inositol , 0.3 mg/l thiamine-HCL, 0.3 mg/l Picloram (PIC), 0.1 mg/l kinetin and 400 mg/l haemoglobin effected a better response with respect to embryogenic callus induction. After 8 weeks of culture, a high frequency of embryoid induction was observed on medium containing MS basal salts, 100 mg/l myo-inositol, 0.3 mg/l PIC , 0.1 mg/l isopentenyl adenine, 1.0 g/l NH₄NO₃ and 400 mg/l haemoglobin. Plant regeneration was observed in 75.8% of the mature somatic embryos, and whole plant regeneration was achieved within 6–7 months of culture. The regenerated plantlets were fertile and similar to in vivo-grown, seed-derived plants except that they were phenotypically smaller. A positive influence of haemoglobin was observed at concentrations up to 400 mg/l at all stages of somatic embryogenesis. The increase in the levels of antioxidant enzyme activities, for example superoxide dismutase and peroxidase, indicated the presence of excess oxygen uptake and the stressed condition of the plant tissues that arose from haemoglobin supplementation. This increased oxygen uptake and haemoglobin-mediated stress appeared to accelerate somatic embryogenesis in cotton.Abbreviations BAP Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA₃ Gibberellic acid - GR Glutathione reductase - 2iP Isopentenyl adenine - KT Kinetin - NAA Naphthaleneacetic acid - PFC Perfluorocarbon - PIC Picloram - PO Peroxidase - ROS Reactive oxygen species - SOD Superoxide dismutase - T.HCl Thiamine hydrochloride     

Factors influencing <Emphasis Type="Italic">in vitro</Emphasis> regeneration from protoplasts of wild cotton (<Emphasis Type="Italic">G. klotzschianum</Emphasis> A) and RAPD analysis of regenerated plantlets

Plants of a diploid wild cotton species (G. klotzschianum A.) were efficiently regenerated from protoplasts isolated from immature somatic embryos and suspension cultures by studying various factors affecting regeneration. Purified protoplasts were cultured with the density of 2–10×10⁵ ml⁻¹, and the medium was k₃ inorganic salts with modified KM₈P organic compositions, supplemented with several combinations of PGRs. Calluses were formed from protoplasts of suspension cultures and immature somatic embryos. The influences of carbon sources and GA₃ on callus differentiation and somatic embryo germination were analyzed. Somatic embryos germinated normally and formed regenerated plantlets. Regenerated plantlets were transferred to the soil and seeds were obtained. Random amplified polymorphic DNA (RAPD) analysis using 80 arbitrary oligonucleotide 10-mers showed 23 primers that gave 74 clear reproducible bands, with amplification products being monomorphic for 14 tested plantlets. A total of 1036 bands obtained exhibited no aberration in RAPD banding patterns in the 14 plants. Plants regenerated via somatic embryogenesis from the diploid cotton protoplasts have genetic homogeneity.     

Plant regeneration via somatic embryogenesis in cotton

An efficient in vitro plant regeneration system characterized by rapid and continuous production of somatic embryos using leaf and stem explants of abnormal seedling as an explant have been developed in Gossypium hirsutum L. Embryogenic callus and somatic embryos have been obtained directly from the explants of cotton abnormal seedlings. Plant growth regulators influenced the induction of cotton somatic embryogenesis. The optimal medium for direct somatic embryogenesis was modified MS medium supplemented with 0.1 mg l^-1 ZT and 2 g l^-1 activated carbon. On this medium, an average of 28.0 and 28.1 matured somatic embryos formed from per leaf and stem explants respectively. The highest frequency of somatic embryogenesis was 100%. The somatic embryos were converted into normal plantlets when cultured on modified MS medium supplemented with 0.1 mg l^-1 ZT. Upon transfer to soil, plants grew well and appeared normal. Plants could be regenerated within 60–80 days. The system of cotton somatic embryogenesis and plant regeneration described here will facilitate the application of plant tissue culture and genetic engineering on cotton genetic improvement. This revised version was published online in June 2006 with corrections to the Cover Date.     

一种新型的棉花体细胞胚胎发生的快速诱导法

用异常苗的茎段和叶片进行培养,可快速高效诱导获得棉花体细胞胚胎发生,激素组膈及其浓度配比影响异常苗的直接胚胎发生,在附加有０．１ｍｇ／Ｌ ＩＡＡ和０．１ｍｇ／ＬＺＴ的改良ＭＳ培养基上,异常苗不仅体细胞胚胎发生率高,而且形成的体细胞胚数目多,用异常苗作外植体获得胚性愈伤组织仅需要时间１０ｄ,获得成熟胚需要２０－３０ｄ,获得再生植株需要６０ｄ,大大短于常规方法获得胚性愈伤组织、体细胞胚和再生植株的时间     

Isolation of putative glycoprotein gene from early somatic embryo of carrot and its possible involvement in somatic embryo development

Somatic embryogenesis is a unique process in plant cells. For example, embryogenic cells (EC) of carrot (Daucus carota) maintained in a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) regenerate whole plants via somatic embryogenesis after the depletion of 2,4-D. Although some genes such as C-ABI3 and C-LEC1 have been found to be involved in somatic embryogenesis, the critical molecular and cellular mechanisms for somatic embryogenesis are unknown. To characterize the early mechanism in the induction of somatic embryogenesis, we isolated genes expressed during the early stage of somatic embryogenesis after 2,4-D depletion. Subtractive hybridization screening and subsequent RNA gel blot analysis suggested a candidate gene, Carrot Early Somatic Embryogenesis 1 (C-ESE1). C-ESE1 encodes a protein that has agglutinin and S-locus-glycoprotein domains and its expression is highly specific to primordial cells of somatic embryo. Transgenic carrot cells with reduced expression of C-ESE1 had wide intercellular space and decreased polysaccharides on the cell surface and showed delayed development in somatic embryogenesis. The importance of cell-to-cell attachment in somatic embryogenesis is discussed.     

Somatic embryogenesis, scanning electron microscopy, histology and biochemical analysis at different developing stages of embryogenesis in six date palm (Phoenix dactylifera L.) cultivars

An efficient somatic embryogenesis system has been established in six date palm (Phoenix dactylifera L.) cultivars (Barhee, Zardai, Khalasah, Muzati, Shishi and Zart). Somatic embryogenesis (SE) was growth regulators and cultivars dependent. Friable embryogenic callus was induced from excised shoot tips on MS medium supplemented with various auxins particularly 2,4-dichlorophenoxyacetic acid (2,4-D, 1.5 mg 1^−l). Suspension culture increased embryogenesis potentiality. Only a-naphthaleneacetic acid (NAA, 0.5 mg 1⁻¹) produced somatic embryos in culture. Somatic embryos germinated and converted into plantlets in N⁶-benzyladenine (BAP, 0.75 mg 1^−l) added medium following a treatment with thidiazuron (TDZ, 1.0 mg 1^−l) for maturation. Scanning electron microscopy showed early stages of somatic embryo particularly, globular types, and was in masses. Different developing stages of embryogenesis (heart, torpedo and cotyledonary) were observed under histological preparation of embryogenic callus. Biochemical screening at various stages of somatic embryogenesis (embryogenic callus, somatic embryos, matured, germinated embryos and converted plantlets) of date palm cultivars has been conducted and discussed in detail. The result discussed in this paper indicates that somatic embryos were produced in numbers and converted plantlets can be used as a good source of alternative propagation. Genetic modification to the embryo precursor cell may improve the fruit quality and yield further.     

Calcium increases the yield of somatic embryos in carrot embryogenic suspension cultures

An upward shift in the concentration of calcium present in the medium during somatic embryogenesis increased the number of embryos produced approximately two-fold. This was observed when embryogenic suspension cells grown in 2,4-D medium with the normal calcium concentration of 10^–3 M were transferred to hormone-free medium containing 10^–2 M calcium and when embryogenic suspension cells grown in 2,4-D medium containing 10^–4 M calcium were transferred to hormone-free medium with 10^–3 M calcium. At calcium concentrations between 6·10^–3 and 10^–2 M globular stage somatic embryos were found in cultures supplemented with 2·10^–6 M of 2,4-D indicating that elevated calcium counteracts the inhibitory effect of 2,4-D on somatic embryogenesis. No qualitative changes were found in the pattern of extracellular polypeptides as a result of growth and embryogenesis in media with different calcium concentrations.     

Regeneration of Gossypium hirsutum and G. barbadense from shoot apex tissues for transformation

A method of regenerating cotton plants from the shoot apical meristem of seedlings was developed for use with particle gun and Agrobacterium-mediated transformation. This method was developed to circumvent the problems of genotype restriction and chromosomal damage frequently encountered in cotton regeneration in tissue culture through somatic embryogenesis. In this procedure, the cells of the shoot meristem are targeted for transformation. Normal and fertile plants of Gossypium barbadense Pima S-6, and 19 cultivars of G. hirsutum were regenerated using this method. Shoot regeneration from these tissues was direct and relatively rapid. A MS based, hormone-free medium could be used with all the varieties tested.This project was funded by grants from Cotton Incorporated, Nisshinbo Industries, and a grant from the Texas Agricultural Experiment Station to RHS. Texas Agricultural Experiment Station Technical Article TA-25667.     

Factors important for somatic embryogenesis in zygotic embryo explants ofCapsicum annuum L.

We used four cultivars ofCapsicum annuum L.—Sweet Banana, California Wonder, Yolo Wonder, and Ace—to reexamine the critical factors influencing somatic embryogenesis from zygotic embryo explants, as reported in the literature. When we followed the protocol of Buyukalaca and Mavituna (1996), which had induced somatic embryogenesis from mature zygotic embryos of cv. Ace, only callus was formed without embryogenesis from our mature zygotic embryo expiants. Using the procedures of Harini and Lakshmi Sita (1993) and Binzel et al. (1996), with some modifications, we were able to induce somatic embryogenesis in all four cultivars. Rates of conversion were significantly reduced, from 75% and 65% to 40% and 28% in ’Sweet Banana’ and ’California Wonder’, respectively, when the immature zygotic embryo expiants were held on the induction medium for longer than two weeks. Likewise, somatic embryogenesis of ’Yolo Wonder’ was not observed if the induction medium was supplemented with 10% glucose or fructose, or without 10% sucrose. For somatic embryo induction and eventual plantlet conversion in Yolo Wonder’, maltose could adequately replace sucrose. In all four cultivars, somatic embryos were initiated from immature zygotic explants on media with or without coconut water, under both light and dark conditions.     

Somatic embryogenesis and plant regeneration in two wild cotton species belong to G genome

The present work describes the plant regeneration via somatic embryogenesis in two wild cotton species belonging to G genome: Gossypium nelsonii Fryx and Gossypium australe F Muell. The role of plant hormones and carbohydrates was also evaluated for somatic embryogenesis and somatic embryo development. Normal plants were obtained from G. nelsonii Fryx; abnormal plants and somatic embryos were obtained from G. australe F Muell. The best medium for callus induction for these G genome wild cotton species was MSB₅ supplemented with 0.1 mg L⁻¹ KT and 0.1 mg L⁻¹ 2,4-D. For embryogenic callus proliferation, the best medium used was MSB₅ supplemented with 0.2 mg L⁻¹ KT and 0.5 mg L⁻¹ IBA. The medium MSB₅ supplemented with 0.15 mg L⁻¹ KT and 0.5 mg L⁻¹ NAA was used successfully for root initiation and plant growth. In addition, adding CuSO₄ and AgNO₃ in the callus-inducing and proliferation medium resulted in a number of somatic embryos. Glucose and maltose, the carbon sources in somatic culture, were used for callus induction, but maltose worked even better than glucose for proliferation of embryogenic callus and development of somatic embryos.