Genotypic and exogenous factors affecting shoot regeneration from anther callus of linseed (Linum usitatissimum L.)

Summary The objective of this study was to investigate factors affecting the regeneration capacity of linseed anther culture. Four different environmental conditions in a phytotron were tested with regard to their effects on anther donor plants of cv. Hella. Anther response and shoot regeneration from anther callus was maximal when donor plants were grown in a 16 hrs-day at 14°C day/8°C night temperature. Anthers of four linseed genotypes were cultured on different media. Maximum shoot regeneration was achieved when the induced calli were transferred onto a modified N6 medium containing zeatin (1 mg l^-1). Most of the calli regenerated shoots in the second subculture on regeneration media. Shoots were rooted on modified B5 or MS media containing NAA (0.1 mg l^-1). Cytological examinations of incubated anthers and root tips of regenerated plants indicated that the anther calli were derived from microspores.Abbreviations B5 Gamborg's (1975) medium - BAP 6-benzylaminopurine - 2,4D dichlorophenoxyacetic acid - N6 Chu's (1978) medium - NAA -naphthaleneacetic acid - MS Murashige & Skoog's (1962) medium - ZEA zeatin     

Induction and development of adventitious shoots of Atropa baetica as a means of propagation

In vitro propagation of Atropa baetica was established employing axillary buds. Single buds were cultured through a multiple shoot induction phase, rooting phase, and then followed by acclimatization in soil. For multiple shoot induction, Murashige and Skoog (MS) medium with 3% sucrose, supplemented with either 0.75 or 1.25 mg l^-1 of BAP provided the best results with an average of 5.6 shoots per explant after 31 days of culture. Similar results were obtained with higher BAP concentrations (1.75–2.0 mg l^-1); however, these media had a negative effect on the subsequent root induction due to residual BAP effect. Medium containing only 0.25 mg l^-1 of BAP induced a significantly lower number of shoots. Root induction occurred spontaneously after transferring the shoots onto MS medium lacking any plant growth regulator. Moreover, root induction also occurred on media supplemented with 0.125 and 0.25 mg l^-1 of NAA. On these two rooting media, this response was more prominent and with a higher number of roots per explant. Nevertheless, after 28 days on root induction medium, the number of rooted plantlets was similar on the three media. Acclimatization of plantlets in soil was very successful (95.52%). However, all plantlets which died during acclimatization were rooted on medium containing 0.25 mg l^-1 NAA suggesting a negative carry over effect of this medium upon plantlet survival, irrespective of the initial BAP treatment used. On the other hand, karyological studies showed no variation in the number of chromosome (2n=72) in root tips of the plantlets produced. This revised version was published online in July 2006 with corrections to the Cover Date.     

High frequency direct plant regeneration,micropropagation and Shikonin induction in <Emphasis Type="Italic">Arnebia hispidissima</Emphasis>

The data presented herein reports a rapid and efficient method for direct plant regeneration at high frequency without intervening callus formation from shoot tip (93%) and nodal segment (60%) cultured on MS media supplemented with 0.5 mg l⁻¹ KIN, 0.25 mg l⁻¹ BAP, 0.1 mg l⁻¹ IAA and 100 mg l⁻¹ CH. Conversely, leaf and internodal explants were poorly responsive. Adventitious shoot buds arose not only from the cut ends but all along the surface of the explants leading to the formation of clusters with multiple shoots. Multiple shoots upon transfer to MS media supplemented with 2.0 mg l⁻¹ IBA induced efficient rooting (80%). In vitro flowering was observed when tissue culture-raised plantlets were maintained for extended period in culture. Shikonin was induced in roots of regenerated plants which often exudates in the culture medium was quantified spectrophotometerically by recording absorbance at 620 nm and estimated to be 0.50 mg g⁻¹ fresh weight of tissue at the end of the 50 days of culture. The regenerated plants were successfully acclimatized, hardened, and transferred to soil in green house for micropropagation. The protocol developed here will be very useful for the supply of Arnebia hispidissima all year as a raw product necessary for obtaining Shikonin for the cosmetic, dyeing, food, and pharmaceutical industries.     

High frequency plantlet regeneration from nodal segment culture of female <Emphasis Type="Italic">Momordica dioica</Emphasis> (Roxb.)

An in vitro propagation method for female plants of Momordica dioica (Roxb.) has been established. The nodal segments were harvested and the cut ends of the explants were sealed with wax and then surface sterilized and cultured. Bud breaking occurred on Murashige and Skoog’s (MS) agar-gelled medium + 2.0 mg L⁻¹ 6-Benzylaminopurine (BAP) + 0.1 mg L⁻¹ Indole-3 acetic acid (IAA). The cultures were amplified by passages on MS medium supplemented with 1.0 mg L⁻¹ BAP + 0.1 mg L⁻¹ IAA. Further, shoot amplification (29.2 shoots per vessel) was achieved by subculturing of in vitro regenerated shoot clump on MS medium + 0.5 mg L⁻¹ BAP + 0.1 mg L⁻¹ IAA. The micropropagated shoots were subsequently transferred for root formation on half-strength MS medium + 2.0 mg L⁻¹ Indole-3 butyric acid (IBA) with 89% success rate. The in vitro-regenerated shoots were also rooted ex vitro with 34% success. These plantlets were hardened in the greenhouse and transferred to the field. The established protocol is suitable for true to type cloning of mature female plant of M. dioica.     

Intergeneric hybridization between Brassica species and Crambe abyssinica

A protocol for high frequency callus induction and plant regeneration from sunflower (Helianthus annuus L.) anthers is described. Different variables using Murashige & Skoog (MS) basal medium supplemented with 2.0 mg/l α-naphthaleneacetic acid (NAA) and 1.0 mg/l N₆-benzyladenine (BA) were tested for their ability to enhance the frequency of anther callusing and subsequent embryogenesis. Of these, agar concentration, sucrose concentration, carbohydrate source had significant effect on callusing, while differences due to incubation under dark vs light conditions, cold pretreatment of capitula for 1 to 6 days prior to anther inoculation and genotype on callusing were non-significant. However, all these factors exerted highly significant influence on embryogenesis when calli from the various media were transferred to medium supplemented with 0.1 mg/l NAA and 0.5 mg/l BA. With the procedure developed, callusing as high as 100% and embryo formation at a frequency of 44% was achieved. Although complete embryos were formed the frequency of their conversion to whole plantlets was low (14.3%). Hence, the embryogenic pathway was bypassed to obtain multiple shoots by transferring embryogenic calli with developing embryos to MS medium supplemented with 0.5 mg/l BA. Elongated shoots rooted on half-strength MS medium supplemented with 0.5 mg/l NAA. Cytological analysis of embryogenic callus and somatic embryos revealed haploids at a frequency of 30% while that of rooted plants showed haploid regenerants at a frequency of 8.3%. Nevertheless, the frequency of putative haploid plants could be enhanced through mass multiplication using nodal explants of the regenerants. This revised version was published online in August 2006 with corrections to the Cover Date.     

High frequency direct plant regeneration from leaf and petals of Cape Primrose (<Emphasis Type="Italic">Streptocarpus</Emphasis>)

High frequency direct plant regeneration from leaf and petal explants was accomplished for the first time in Streptocarpus varieties. The shoot induction frequency varied with respect to the benzylaminopurine (BAP) concentration added to the Murashige and Skoog (MS) medium. MS medium with 0.5 mg l⁻¹ BAP exhibited the highest (69.9%) plant regeneration frequency with an average of 186 shoots per explant. A higher concentration of BAP inhibited shoot bud induction and plant regeneration along with necrosis of explants. Petal explants derived from the varieties ‘Branwen’ (pink and white) and ‘Chorus Line’ (violet and white) displayed plant regeneration frequency of 22.2–47.4% (within a total of 12 weeks) on MS medium containing 2.0 mg l⁻¹ α-naphthaleneacetic acid and 0.5 mg l⁻¹ BAP for 8 weeks followed by 4 weeks on MS medium with 1.0 mg l⁻¹ BAP. Scanning electron microscopy confirmed direct plant regeneration without callus. Regenerated plants from leaf explants with well-developed leaves and roots were hardened and successfully transferred to pots in glasshouse exhibiting 86% survival at the end of 4–6 weeks. Whereas, regenerated plants from flower petal explants upon transfer to pots in glasshouse exhibited 75–82% survival at the end of 4–6 weeks.     

Production of intergeneric hybrid plants between <Emphasis Type="Italic">Sandersonia aurantiaca</Emphasis> and <Emphasis Type="Italic">Gloriosa rothschildiana</Emphasis> via ovule culture (Colchicaceae)

Intergeneric hybrid plants between Colchicaceous ornamental plants, Sandersonia aurantiaca and Gloriosa rothschildiana, have successfully been produced via ovule culture. After 5 days of reciprocal cross-pollination, a few pollen tubes were observed in the ovary. Although seeds were obtained in both reciprocal cross-combinations, they did not germinate under ex vitro conditions. Ovules with placental tissues isolated 14 days after cross-pollination of S. aurantiaca × G. rothschildiana were cultured on a medium containing 0.01 mg l^–1 each of -naphthaleneacetic acid (NAA) and 6-benzyladenine (BA), on which 41.5% of ovules swollen and produced callus-like structures within 10 weeks. When such swollen ovules were transferred to a medium containing 0.1 mg l^–1 each of NAA and BA, 7.5% of the initially cultured ovules produced rhizome-like structures within 6 weeks. Among the rhizome-like structures, those derived from two independent ovules (3.7% of the initially cultured ovules) produced multiple shoots following transfer to a medium containing 0.25 mg l^–1 NAA and 2.5 mg l^–1 BA. Multiple shoot-derived plantlets were established on a plant growth regulator-free medium, and they were successfully transplanted to pots. Early verification of their hybridity was accomplished by flow cytometry (FCM) analysis, chromosome observation and rDNA analysis.     

In vitro culture of immature seed for rapid generation advancement in tomato

The importance of fast-trackt generation advancement in developing superior germplasm has been recognized in breeding of many crop species. To address this issue in tomato, immature seeds were excised from fruit at different maturity stages and transferred to culture medium. The best culture medium was modified full strength Moorashige–Skoog (MS) salts supplemented with 0.1 mg l⁻¹ IAA, 0.5 mg l⁻¹ IBA, 0.5 mg l⁻¹ GA₃ and 2% sucrose. If the excised seeds were able to grow, most showed shoot formation after a week. Seeds extracted as early as 10 days after pollination were successfully cultured provided they were transferred aseptically and without injury. No morphological or physiological changes in regenerated plants and their fruit relative to the parent were detected. Germination from immature seeds of tomato is a simpler alternative to in vitro culture of immature embryos or callus, as it can be undertaken in comparatively less stringent laboratory conditions. Using this approach, five generations can be produced in a year in contrast to a maximum of three generations with conventional methods. This offers an opportunity for rapid generation advancement aimed towards population development when coupled with marker assisted selection in tomato breeding for biotic and abiotic stress tolerance.     

天蓝苜蓿花药愈伤组织诱导及再生体系的建立

为获得天蓝苜蓿单倍体再生植株,本研究以野生天蓝苜蓿花药为外植体,采用正交设计L₁₆(4⁴)筛选适宜天蓝苜蓿愈伤组织诱导培养基,比较不同基本培养基及生长调节剂组合筛选适宜的分化培养基,并用1/3MS、1/2MS和MS添加不同浓度的NAA研究不定生根。结果表明:天蓝苜蓿现蕾15~25 d的花药其愈伤组织诱导效果最好,高达60.5%。4℃低温预处理2~4 d有利于愈伤组织诱导,诱导率达77.2%。适宜花药愈伤组织诱导的培养基为NB+2,4-D 1.0 mg/L+NAA 0.5 mg/L+6-BA 0.5 mg/L+TDZ 1.0 mg/L,诱导率达78.5%。比较不同基本培养及生长调节剂的不同组合发现,NB培养基愈伤组织分化效果优于B₅和MS,NB+NAA0.5 mg/L+6-BA 2.0 mg/L适宜愈伤组织的分化,分化率为66.3%。不定芽在1/3MS+NAA 0.5 mg/L中培养,生根率最高,为86.55%。本研究建立了天蓝苜蓿花药培养再生体系,获得了单倍体植株,为天蓝苜蓿的育种实践及基因组学研究提供基础材料。     

Resynthesizing <Emphasis Type="Italic">Brassica napus</Emphasis> from interspecific hybridization between <Emphasis Type="Italic">Brassica rapa</Emphasis> and <Emphasis Type="Italic">B. oleracea</Emphasis> through ovary culture

Using three varieties of Brassica rapa, cv. Hauarad (accession 708), cv. Maoshan-3 (714) and cv. Youbai (715), as the maternal plants and one variety of B. oleracea cv. Jingfeng-1 (6012) as the paternal plant, crosses were made to produce interspecific hybrids through ovary culture techniques. A better response of seed formation was observed when ovaries were cultured in vitro at 9–12 days after pollination on the basal MS and B5 media supplemented with 6-benzylaminopurine (BA) and naphthylacetic acid (NAA). The best response was observed for cross 714×6012 with the rate of seeds per ovary reaching 43.0%. Seeds for cross 715×6012 showed the best germination response (66.7%) on the regeneration medium (MS+1.0 mg l^–1 BA+0.05 mg l^–1 NAA). In all three cross combinations, good response in terms of root number and length of plants was observed on the root induction medium (MS+1.0 mg l^–1 BA+0.1 mg l^–1 NAA). A better response was observed for the regenerated plants cultured for 14 days than for 7 days. The ovary-derived plants with well-developed root system were hardened for 8 days and their survival rate reached over 80%. Cytological studies showed that the chromosome number of all plants tested was 19 (the sum of both parents), indicating that these regenerated plants were all true hybrids of B. rapa (n = 10) × B. oleracea (n = 9). The regenerated plants were doubled with colchicine treatment, and the best response in the crosses 708×6012, 714×6012 and 715×6012 was observed when treated with 170 mg l^–1 colchicine for up to 30 h and their doubling frequency reached 52, 56 and 62%, respectively.     

Corms as a source of explants for the successful clonal propagation of <Emphasis Type="Italic">Crocus cancellatus</Emphasis>

The genus Crocus comprises plants with a potential to be developed as a new ornamental crop but to date, there are not many reports on in vitro propagation of many members of this genus. The present study involves in vitro propagation of Crocus cancellatus with ornamental and horticultural value. Two different types of corm explants (apical and basal halves of corms) were cultivated onto Murashige and Skoog’s (MS) medium supplemented with different levels of α-naphthalene acetic acid (NAA) and 6-benzylaminopurine (BAP). One to five cormlets emerged from every responding explant through direct organogenesis. Apical halves of corms were more highly responsive than basal halves and produced a maximum multiplication rate with 3.45 ± 0.06 cormlets per explant in 95.33 ± 2.33% of the explants in MS medium supplemented with 3% sucrose and 2 mg L⁻¹ NAA and 1 mg L⁻¹ BAP. The effect of cold storage temperature on in vitro cormlets sprouting was studied. Cormlets stored at 4°C for 8 weeks had more statistically significant positive effects on cormlets sprouting from the controls. In vitro rooting of cormlets was induced on MS medium without plant hormones.     

日本晚樱组培快繁技术研究

进行了日本晚樱的组培快繁试验。试验结果表明，日本晚樱最佳的增殖培养基是MS/ML + 6-BA 0.5 mg/L + NAA0.05 mg/L +白糖40g/L +琼脂4.5g/L,增殖系数为2.47~2.56；最佳生根培养基为ML+IBA0.05mg/L +NAA0.05 mg/L +白糖20g/L +琼脂4.5g/L，生根率为100%；炼苗12~17d后移栽至草炭土中，成活率达90%。     

Anther culture of recalcitrant indica × Basmati rice hybrids

Fertile, green, di-haploid plants were obtained at high frequencies from several indica × Basmati rice F₁ hybrids and/or F₂ plant populations using an improved anther culture procedure. Anthers from cold-pretreated (10 ^°C for 10 d) panicles of six indica (HKR120, HKR86-3, HKR86-217, PR106, Gobind andCH2 double dwarf) and two Basmati rice (Basmati 370,Taraori Basmati) varieties and 14 heterotic indica ×Basmati F₁/F₂ hybrids were cultured in modified agarose-solidified N6M, Heh5M and RZM media. Best callus induction frequencies (2.6–78%) were obtained in RZM medium containing 4% (w/v) maltose,2,4-D, NAA and kinetin. F₂ plants compared to F₁ hybrids and parental rice varieties, were more responsive to anther culture. Androgenesis frequencies of 31–78% were obtained for indica × Basmati F₂ plants in RZM medium in just 30 d which are comparable to or higher than that reported for japonica rice varieties and hybrids involving japonica rice parent(s). Agarose (1.0% w/v)-solidified MS medium containing 3.0% maltose, kinetin, BAP, and NAA, induced green shoot regeneration in 0–51% of the anther-derived callide pending upon the genotype. High plant regeneration frequencies (67–337 green plants per 1000 anthers)were obtained from anther calli of several F₁hybrids (Gobind × Basmati 370 and HKR120 ×Taraori Basmati) and F₂ plants (Gobind × Basmati370, Gobind × Taraori Basmati, HKR86-3 × TaraoriBasmati). A sample of 498 plants obtained from the above hybrids, were transferred to pots with>90% survival; 8–78% of these plants had >5%spikelet fertility and were diploid. In addition,18% of the haploid plants could be diploidized by submerging in 0.1% colchicine solution for 16–18 h. The improved anther culture procedure reported here, resulted in several fold increase in the recovery of green plants from recalcitrant indica × Basmati rice hybrids compared to previous published procedures. The study may accelerate the introgression of desirable genes from indica into Basmati rice using anther culture as a breeding tool. This revised version was published online in July 2006 with corrections to the Cover Date.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of pigeon pea [<Emphasis Type="Italic">Cajanus cajan</Emphasis> (L.) Millsp.] for resistance to legume pod borer <Emphasis Type="Italic">Helicoverpa armigera</Emphasis>

Agrobacterium-mediated genetic transformation was performed using embryonic axes explants of pigeon pea. Both legume pod borer resistant gene (cry1Ac) and plant selectable marker neomycine phosphor transferase (nptII) genes under the constitutive expression of the cauliflower mosaic virus 35S promoter (CaMV35S) assembled in pPZP211 binary vector were used for the experiments. An optimum average of 44.61% successfully hardened dot blot Southern hybridization positive plants were obtained on co-cultivation media supplemented with 200 μM acetosyringone without L-cysteine. The increased transformation efficiency from a baseline of 11.53% without acetosyringone to 44.61% with acetosyringone was further declined with the addition of different concentrations of L-cysteine to co-cultivation media. Transgenic shoots were selected on 50 and 75 mg L⁻¹ kanamycin. Rooting efficiency was 100% on half-strength Murashige and Skoog medium with 20 g L⁻¹ sucrose and 0.5 mg L⁻¹ indole butyric acid in the absence of kanamycin. Furthermore, 100% seed setting was found among all the transgenic events. The plants obtained were subjected to multi- and nochoice tests to determine the behavioral responses and mortality through Helicoverpa armigera bioassays on the leaf and relate their relationship with the expression of cry1Ac protein which was found to be less in leaf as compared to the floral buds, anther, pod, and seed.     

组织培养快速繁殖龙翅海棠

龙翅海棠的组培可以幼嫩叶片作外植体,通过诱导形成愈伤组织分化不定芽的途径,建立无菌系,获得再生植株.最适诱导分化培养基为Ms 1mg/L6-BA 0.1 mg/L NAA 3%蔗糖 0.6%琼脂;最适增殖培养基为:Ms 0.8mg/L6-BA 0.08mg/L NAA 3%蔗糖 0.6%琼脂;最适生根培养基为:1/2MS 0.08mg/L NAA 0.02mg/LIBA 3%蔗糖 0.6%琼脂;最适移栽基质为河沙∶珍珠岩=2∶1.增殖周期为35d.     

Production of two mandarin × trifoliate orange hybrid populations via embryo rescue with verification by SSR analysis

Intergeneric sexual hybridizations were conducted between two genera of Rutaceae, with Satsuma mandarin (Citrus unshiu Marc) and Red tangerine (C. reticulata Blanco) as maternal parents, and Poncirus trifoliata (L.) Raf as the paternal parent, in an effort to generate hybrid populations for both molecular mapping and rootstock breeding. Embryo rescue is important for citrus sexual breeding because polyembryony can interfere with hybrid embryo recovery. Immature embryos of 80, 85, 90 days after pollination (DAP) from the Satsuma mandarin (S) × trifoliate orange (P) cross, 80 and 85 DAP from the Red tangerine (R) × trifoliate orange (P) cross, were cultured on MG1.0 medium consisting of MT basal medium supplemented with 1.0 mg l⁻¹ GA₃ and 4% sucrose. The results showed that 80 DAP was the optimal time for embryo rescue of the tested crosses, as evidenced by embryos at this stage exhibiting high germination rates, 37.3% for S × P and 51.3% for R × P. Among the eight tested media, MT medium supplemented with 0.5 mg l⁻¹ GA₃ was the best one for Satsuma mandarin, and MT plus 1.0 mg l⁻¹ GA₃ for Red tangerine. A total of 85 plants were obtained from S × P, and 340 from R × P. Out of them, 44 progenies from S × P and 111 from R × P were sufficiently confirmed to be hybrids by morphological characterization and SSR analysis. In addition, two hybrid callus lines were obtained from S × P and R × P respectively.     

An efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation protocol for black pepper (<Emphasis Type="Italic">Piper nigrum</Emphasis> L.) using embryogenic mass as explant

A protocol was developed for an efficient Agrobactertium-mediated transformation of black pepper plants through somatic embryogenesis. Embryogenic mass derived from primary somatic embryos that were obtained from the micropylar region of mature germinating seeds of black pepper was found to be the ideal target tissue for transformation. Genetic fidelity test of embryogenic mass-derived plantlets by RAPD using 23 random primers revealed no genetic variation among the progenies and the parent plant. Among the antibiotics used for selection of transformants, cefotaxime at 100 μg mL⁻¹ was found to be optimum to control Agrobacterium besides its ability to promote somatic embryo proliferation. In the case of kanamycin, a step-wise increase in concentration from 25 to 50 and then to 100 μg mL⁻¹ were found to be optimum. Embryogenic mass co-cultivated with Agrobacterium carrying the β-glucuronidase (GUS) reporter gene were cultured on plant growth regulator-free Schenk and Hildebrandt (SH) medium and transformants were selected in selection medium containing cefotaxime and step-wise increase in kanamycin concentration. The transient GUS gene expression was determined histochemically. Transformants that survived in the selection medium were hardened in the greenhouse. An average of nine hardened putative plantlets was obtained per gram of embryogenic mass. The presence of transgene in these plantlets was assayed by PCR, dot blot, and Southern blot hybridization. Results presented demonstrated for the first time an efficient transformation and regeneration of black pepper without the use of growth regulators. This simple efficient procedure would allow transformation of black pepper with genes of desirable characters.     

Comparison of agar and gum karaya as gelling agent for in vitro regeneration of rough lemon (Citrus jambhiri Lush.) plantlets from nodal explants

In the present study, attempt was made to compare agar with gum karaya as gelling agent in micropropagation of rough lemon (Citrus jambhiri Lush.). Initially nodal segments were cultured on agar-gel MS medium containing benzyladenine (BA), kinetin (KN), zeatin (ZN) (1.0–2.5 mg L^?1) and malt extract (200–1,200 mg L^?1) to standardize the medium. Maximum shoot regeneration (66.66%) was observed with KN 2 mg L^?1 with an average shoot length of 0.73 cm. Gum karaya and agar was then evaluated at different concentration and combinations in same medium. The shoot regeneration response on media gelled with 30 g L^?1 gum karaya was 62.49% with an average shoot length of 0.80 cm. Regenerated shoots were rooted on MS medium gelled with agar and supplemented with different concentrations (0.5–2.5 mg L^?1) of indole-3-acetic acid (IAA), naphthalene acetic acid (NAA), and indole-3-butyric acid (IBA). Maximum response (52.77 %) was observed with IBA 2.0 mg L^?1 with an average number of 2.58 roots/shoot. A maximum of 53.47% cultures showed root regeneration with an average number of 2.91 roots/shoot in 30 g L^?1 gum karaya-gel medium. Texture measurements revealed that firmness of gum karaya-gel medium was nowhere near to that of agar. However, in their capability of supporting growth and differentiation of explants they are equal to agar medium. Gum karaya forms less adhesive and gummy medium as compared to agar. This study indicates that gum karaya can be used as gelling agent in place of agar.     

Foliar application of the exogenous plant hormones at pre-blooming stage improves flowering and fruiting in cashew (<Emphasis Type="Italic">Anacardium occidentale</Emphasis> L.)

The problem of declining tree yield has led to an investigation into the effectiveness of foliar application of exogenous hormones to improve flowering, fruit set, and fruit retention in cashew. Five exogenous hormones, one Gibberellic Acid (GA₃) and four Auxins (IAA, IBA, NAA, and 2,4-D) at seven different rates of application (0 mg L⁻¹, 10 mg L⁻¹, 25 mg L⁻¹, 50 mg L⁻¹, 100 mg L⁻¹, 250 mg L⁻¹, and 500 mg L⁻¹) were tested on six yield-related components of the two Brazilian cashew genotypes. This trial was a factorial split-split-plot design with each treatment replicated five times within a tree and three replications (three trees) per genotype. Responses varied significantly between exogenous hormones, concentrations and genotypes. The cashew plants used showed hormone-specific and optimum concentration response patterns. Of the five exogenous hormones tested, GA₃ was most effective as its application at 50–100 mg L⁻¹ gave five-fold improvements in flowering (precocity and number of hermaphrodite flowers) and fruiting, and about 69% increase in fruit retention ability and 25% in nut size. Panicles treated with GA₃ also produced relatively bigger nuts compared to the untreated. Days to flowering was found to be hormone sensitive, while production of hermaphrodite flowers, fruit set, and nut development tended to be concentration specific. The GA₃ exhibited a broad concentration tolerance among the five exogenous hormones investigated. Our data showed that using GA₃ at 50 mg L⁻¹ will enhance flowering precocity, shorten flowering duration, increase production of hermaphrodite flowers and fruit set significantly, and resultant nuts develop optimally with high percentage retention. Thus, it suggests cashew yield could be increased by exogenous foliar application of GA₃ at 50–100 mg L⁻¹ at pre-blooming stage.     

Continuous Plant Regeneration from Established Embryogenic Cell Suspension Cultures of Italian Ryegrass and Tall Fescue

The suitability of different protocols was compared for entire plant regeneration by somatic embryogenesis, of the forage plants Lolium multiflorum Lam. (Italian ryegrass) and Festuca arundinacea Schreb. (tall fescue). In the first protocol, miniature embryos were used as starting material, while mature seeds were retained in the other two. Whichever the considered protocol, undifferentiated calli were produced on Murashige and Skoog MS medium supplemented with 2,4-D. The calli were subcultured in the dark on solid MS agar medium, containing 5 mg/1 2,4-D (protocol 2) or on solid MS medium followed by transfer to a rotated liquid MS medium with 2 mg/1 2,4-D (protocol 1). In these conditions, induction of somatic embryogenesis occurred, and whole plants were regenerated during a limited lapse of time, upon transfer in the light, to MS medium supplemented with BAP but devoid of 2,4-D. The simultaneous elimination of 2,4-D and transfer to light appeared essential for full regeneration of the plants. Using this characteristic, an additional step was added to a new protocol (protocol 3) in which microcalli, cultured on liquid MS medium containing 5 mg/1 2,4-D, were transferred to the same medium with 2 mg/1 2,4-D, in the dark. In these conditions, the suspensions kept their embryogenic potential for months. In all cases, plantlets were successfully transferred into the soil. An evaluation of the somaclonal variation potential of the plants issued from each protocol is now underway.