Potential,constraints and applications of in vitro methods in improving grain legumes

Grain legumes, the important constituents of sustainability‐based cropping systems and energy‐limited vegetarian diets have long been the subject of scientific research. Tremendous technological strides were made in the so‐called orphan crops, in terms of both varietal improvement and generation of basic information. Despite recalcitrancy and high genotype dependency, in vitro culture techniques such as organogenesis, in vitro mutagenesis, embryo rescue and in vitro gene transfer have been deployed for improvement of several grain legumes and these played an important role in introgression of desirable genes from related and distant species and creation of additional genetic variability. Stable and reproducible regeneration protocols resulted in the development of genetically modified chickpea, pigeon pea, cowpea, mungbean, etc., while embryo rescue was deployed successfully for recovery of interspecific recombinants, a few of them exploited for the development of commercial cultivars. Nevertheless, doubled haploidy witnessed limited success and protoplast regeneration and in vitro mutagenesis remained of academic interest. The present review focuses on the progress, achievements, constraints and perspectives of using in vitro technology in grain legume improvement.     

A Review on Regeneration in Cowpea （Vigna unguiculata L. Walp）

Vigna unguiculata L. Walp is a recalcitrant plant in terms of in vitro cell, tissue and organ differentiation, which makes it difficult to apply tissue-culture dependant approaches for obtaining stable genetic transformation in cowpea. Despite this, sporadic efforts have been made to develop regeneration systems in cowpea during the past 40 years. This review presents the considerable progress on cowpea regeneration （organogenesis and embryogenesis） and especially focuses on the regeneration mode of organogenesis, including highlights of the effect of genotypes, explants, medium and plant hormones used in tissue culture. The existing problems and the future research directions were also discussed.     

Bulb organogenesis of Tulipa tarda in vitro cultures in relation to light environment

Here organogenesis of tarda tulip (Tulipa tarda Stapf.) from callus explants is presented. The callus tissue was cultivated on MS media containing 3% or 6% sucrose and either no addition of BAP (6-benzyl-aminopurine) or supplemented with 0.5 μM BAP. The cultures were maintained under a 16?h photoperiod under white, red or blue fluorescent light, at 20?±?2°C for 12 weeks. This study aimed to determine the most suitable light conditions and medium composition for seed-derived callus explants in order to obtain an efficient formation of adventitious bulbs. There were no differences between the spectra of light in differentiating adventitious bulbs. Explants cultured in darkness (control), on 0.5?µM BAP and 3% sucrose, formed the highest number of adventitious bulbs. The efficiency of adventitious organogenesis amounted to 36.6 bulbs per 1?g of callus tissue. The fresh weight of biomass, cultured in these conditions, increased within 12 weeks from 1 to 6.99?g. Supplementation with BAP of the medium containing 3% sucrose promoted the formation of bulbs, but in the case of the medium with 6% sucrose, BAP had an adverse influence under every type of light. The obtained results provide a useful protocol for the micropropagation of T. tarda, which can be used commercially for rapid and cost-effective production of the tulip.     

Genotypic differences in organogenesis from callus of ten triticale lines

Summary Callus was obtained from immature excised embryos of triticale using MS medium supplemented with 3 mg/l 2,4-D and 1 mg/l kinetin. The presence of 2,4-D was essential for continued callus proliferation. Plantlets were induced from the calli by sub-culturing on medium either devoid of auxin or containing 0.1 mg/l 2,4-D. The capacity to produce callus and to form organs and plantlets differed markedly among the genotypes used. Lines also had distinct response to presence and absence of 2,4-D in the regeneration media. The callus of most triticale lines used differentiated into organs more readily on MS medium supplemented with 0.1 mg/l 2,4-D than on medium without growth regulators. Very high frequencies (up to 75%) of plantlet regeneration were observed in several of the triticale lines studied.     

杂交鹅掌楸离体培养中器官发生的研究

以杂交鹅掌楸的叶片、叶柄、无菌苗的茎段及其芽基部切段为外植体,进行愈伤组织途径的器官发生及不定芽途径的直接器官发生培养。结果表明,多种外植体在诱导培养基上均能脱分化产生愈伤组织,其中无菌苗芽基部具有最高的愈伤组织诱导率。愈伤组织在MS 6-BA 2.0 mg.L-1 NAA 0.5 mg.L-1和MS 6-BA 4.0 mg.L-1 NAA 0.5 mg.L-1的分化培养基上能分化出不定芽。部分外植体在MS 6-BA 4.0 mg.L-1 NAA 0.5 mg.L-1的分化培养基上直接分化产生不定芽。器官发生途径再生体系的建立为抗逆基因工程等工作奠定了基础。     

文心兰花梗外植体形态建成初步研究

通过对文心兰花梗外植体的诱导培养及其形态建成途径观察研究发现,外植体通过不定芽（器官型）和愈伤组织成芽（器官发生型）以及PLB途径,最终发育形成完整植株。     

A comparison of the somaclonal variation level of Rosa hybrida L. cv Meirutral plants regenerated from callus or direct induction from different vegetative and embryonic tissues

Summary Flowering plants of Rosa hybrida L. cv Meirutral have been obtained either from direct regeneration of adventitious shoots on leaf and root fragments, or through organogenesis and somatic embryogenesis on calli derived from anther, ovule, petal, sepal, receptacle, leaf, stem internode, root and zygotic embryo tissues. The calli derived from floral parts exhibited rhizogenesis. In this case direct induction of adventitious shoots from selected roots had to be performed in order to generate plants. A histological study of the morphogenetic calli was carried out. The plants regenerated directly and those regenerated from calli of leaf, stem internode, root and zygotic embryo tissues, together with reference plants propagated by cuttings, were compared on a phenotypic basis by taking into account petal number, form and colour, and plant growth habit. From these observations, it can be concluded that directly regenerated plants are as stable as reference plants while plants regenerated from callus are unstable, especially those derived from zygotic embryo tissues.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA3 gibberellic acid - IAA 3-indole-acetic acid - MS Murashige and Skoog - NAA 1-naphthalene acetic acid     

Relationship between somaclonal variation and type of culture in cucumber

Highly inbred B line of cucumber was used to compare the effect of four types of in vitro culture on somaclonal variation. The plants were regenerated from the following types of culture: twelve- and eighteen-month-old liquid culture of meristematic clumps (LMC₁₂₍₁₈₎), ten-month-old embryogenic cytokinin-dependent suspension (CDS), eighteen-month-old embryogenic cytokinin-dependent suspension in medium with modified NH⁺ ₄/NO₃ ^- ratio (CDS 1.7), twelve-month-old embryogenic auxin-dependent suspension (ADS), thirty six-month-old embryogenic auxin-dependent suspension in medium with modified NH⁺ ₄/NO₃ ^- ratio (ADS 1.7) and recurrent leaf callus regeneration (RLC) – repeated 5 times. The differences in the incidence of the following properties were observed: the ploidy of R₀ plants, the segregation of new morphological traits in R₁ and the germination ability of R₁ seeds. R₁ families with the segregation of new phenotypes were most numerous in CDS (62.5%) and LMC₁₈ (57.9%), next in CDS1.7 (35.7%), while the smallest number was found in LMC₁₂ (11.1%) and RLC (3.4%).Tetraploid and mixoploid plants occurred in ADS1.7 and ADS (100%) whereas CDS and RLC were observed to contain only tetraploids, respectively 33.3% and 55.2%. There were no changes of ploidy after LMC₁₂, LMC₁₈ and CDS1.7. Among new phenotypes there were such that have not been described so far in cucumber: ginkgolike leaf (gll), yellow-green chlorophyll mutants (y-gc), serrate margin of corolla in male and female flowers (smc). This revised version was published online in August 2006 with corrections to the Cover Date.     

苹果叶片愈伤组织植株再生研究

本研究以苹果品种千秋试管苗叶片为试材，接种子ＭＳ附加不同浓度ＢＡ和ＮＡＡ激素组合的培养基上，经愈伤组织诱导、不定芽诱导，一步诱导千秋叶片再生不定芽。得到苹果品种千秋叶片－愈伤组织－不定芽再生的适宜培养条件为：ＭＳ基本培养基附加ＢＡ ２ｍｇ／Ｌ，ＮＡＡ０．２ｍｇ／Ｌ；黑暗诱导５个星期后在同种培养基中转入光照培养，培养室温度为２５±２℃。     

白檀未成熟胚的器官发生和植株再生

以白檀未成熟胚为试材,以改良MS为培养基,研究了白檀未成熟胚的器官发生和植株再生.结果表明:改良MS(mMS)能够很好的诱导愈伤组织,在添加了0.2 mg/L 6-BA+0.1 mg/L NAA的培养基中,诱导率高达92.5％;胚性愈伤组织分化最佳培养基为mMS+0.25 mg/L 6-BA+0.15 mg/L NAA,分化率为72.4％;分化出的胚性愈伤组织在空白mMS培养基上继代培养15 d,76.6％的组织分化出芽,再转入1/2mMS+ 1.5％蔗糖培养基上培养20 d,芽长至1.5 cm.