D. K. Srivastava

Biotechnological advancement in genetic improvement of broccoli (Brassica oleracea L. var. italica), an important vegetable crop

With the advent of molecular biotechnology, plant genetic engineering techniques have opened an avenue for the genetic improvement of important vegetable crops. Vegetable crop productivity and quality are seriously affected by various biotic and abiotic stresses which destabilize rural economies in many countries. Moreover, absence of proper post-harvest storage and processing facilities leads to qualitative and quantitative losses. In the past four decades, conventional breeding has significantly contributed to the improvement of vegetable yields, quality, post-harvest life, and resistance to biotic and abiotic stresses. However, there are many constraints in conventional breeding, which can only be overcome by advancements made in modern biology. Broccoli (Brassica oleracea L. var. italica) is an important vegetable crop, of the family Brassicaceae; however, various biotic and abiotic stresses cause enormous crop yield losses during the commercial cultivation of broccoli. Thus, genetic engineering can be used as a tool to add specific characteristics to existing cultivars. However, a pre-requisite for transferring genes into plants is the availability of efficient regeneration and transformation techniques. Recent advances in plant genetic engineering provide an opportunity to improve broccoli in many aspects. The goal of this review is to summarize genetic transformation studies on broccoli to draw the attention of researchers and scientists for its further genetic advancement.

Biotechnological applications in in vitro plant regeneration studies of broccoli (Brassica oleracea L. var. italica), an important vegetable crop

Biotechnology holds promise for genetic improvement of important vegetable crops. Broccoli (Brassica oleracea L. var. italica) is an important vegetable crop of the family Brassicaceae. However, various biotic and abiotic stresses cause enormous crop yield losses during commercial cultivation of broccoli. Establishment of a reliable, reproducible and efficient in vitro plant regeneration system with cell and tissue culture is a vital prerequisite for biotechnological application of crop improvement programme. An in vitro plant regeneration technique refers to culturing, cell division, cell multiplication, de-differentiation and differentiation of cells, protoplasts, tissues and organs on defined liquid/solid medium under aseptic and controlled environment. Recent progress in the field of plant tissue culture has made this area one of the most dynamic and promising in experimental biology. There are many published reports on in vitro plant regeneration studies in broccoli including direct organogenesis, indirect organogenesis and somatic embryogenesis. This review summarizes those plant regeneration studies in broccoli that could be helpful in drawing the attention of the researchers and scientists to work on it to produce healthy, biotic and abiotic stress resistant plant material and to carry out genetic transformation studies for the production of transgenic plants.

In vitro plant regeneration studies and their potential applications in Populus spp.: a review

Genus Populus comprises about 25–35 species of deciduous flowering plants in the family Salicaceae which are widely distributed in temperate climates of the Northern Hemisphere. Populus species are important resources in certain branches of industry and have a special role for the scientific study of biological and agricultural systems. The poplar is known for its remarkable significance among the commercially propagated tree species such as teak, eucalyptus, wild cherry, red wood, and radiata pine. In vitro regeneration refers to growing and multiplications of cells, tissues and organs on defined liquid/solid media under aseptic and controlled environments. In vitro clonal propagation of forest trees, due to the high multiplication rate, is an attractive alternative for rapid propagation of elite genotypes of those species that could not easily be propagated through conventional methods. Owing to their widespread uses at the industrial level and for meeting the ever-increasing global demand for biomass p...

Morphogenic Response of Leaf and Petiole Explants of Broccoli Using Thidiazuron

Broccoli (Brassica oleracea var. italica) is an important nutritionally rich vegetable cole crop grown in the world. Environmental stress, pests, and diseases cause enormous yield losses because of a limited gene pool. Genetic manipulation is becoming an important method for broccoli improvement. The objective of present study was to evaluate the potency of thidiazuron (TDZ) as a plant growth regulator in evoking morphogenic responses in leaf and petiole explants of broccoli. An efficient, reproducible, and high frequency plant regeneration protocol has been standardized in broccoli cv. Solan green head. Leaf and petiole explants were cultured on Murashige-Skoog (MS) medium, supplemented with a wide range of TDZ concentrations. The following treatments were designed for efficient in vitro shoot regeneration: TDZ alone, TDZ with adenine, TDZ with naphthalene acetic acid (NAA), and TDZ with indole acetic acid (IAA). Among the 36 combinations of growth regulators used, the highest percentage of leaf explants producing shoot (89.25%) was recorded on MS medium containing 1.0 μM TDZ and 0.107 μM NAA. The multiple shoot regeneration response of petiole explant producing shoots (91.55%) was obtained on MS medium containing 2.0 μM TDZ and 0.107 μM NAA. Shoot multiplication and elongation were obtained on the same medium. For root regeneration in in vitro regenerated shoots, different concentrations of NAA were applied. High frequency (100%) root regeneration response with healthy and vigorous roots was observed on MS medium supplemented with 0.54 μM NAA. The regenerated plantlets with well-developed shoots and root system were transferred to pots containing cocopeat and successfully acclimatized. We recommend 1.0 μM TDZ with 0.107 μM NAA and 2.0 μM TDZ and 0.107 μM NAA combinations for adventitious shoot regeneration from leaf and petiole explants in broccoli cv. Solan green head respectively. This is the first report on high frequency organogenesis from leaf and petiole explants of broccoli cv. Solan green head using thidiazuron.

High frequency regeneration of plants from cotyledon and hypocotyl cultures in Brassica oleracea cv. Pride of India

Highlights • Cabbage “nutrient rich” economically important nutrient rich vegetable crop with secondary metabolites called glucosinolates contributed to anticarcinogenic properties.• Various stress (biotic and abiotic) results in quantitative and qualitative yield losses. So application of plant genetic engineering techniques can be used to cope up with the stresses.• The goal of present investigation was to develop an efficient, reliable and high-frequency plant-regeneration protocol for the introduction of a desirable gene in cabbage, was successfully achieved.

Genetic Engineering for Insect Resistance in Economically Important Vegetable Crops

Vegetables play a vital role in human nutrition and health by providing nutrients, vitamins, antioxidants, phytosterols, and dietary fiber. In the developing world, vegetable farming is a considerable part of the agricultural economy of different nations. Vegetable crop quality and quantity are seriously affected by various biotic and abiotic stresses, which destabilize rural economies in many countries. In the last many decades, conventional breeding has contributed significantly for the improvement of vegetable quality, yields, biotic and abiotic stress resistance, and postharvest management, but there are many constraints in conventional breeding, which can only be overcome by techniques of modern biology for genetic advancements.

Assessment of genetic diversity in lettuce ( Lactuca sativa L.) germplasm using RAPD markers

The importance of germplasm characterization is an important link between the conservation and utilization of plant genetic resources in various breeding programmes. In the present study, genetic variability and relationships among 25 Lactuca sativa L. genotypes were tested using random amplified polymorphic DNA (RAPD) molecular markers. A total of 45 random decamer oligonucleotide primers were examined to generate RAPD profiles, out of these reproducible patterns were obtained with 22 primers. A total of 87 amplicon were obtained, out of which all were polymorphic and 7 were unique bands. The level of polymorphism across genotypes was 100% as revealed by RAPD. Genetic similarity matrix, based on Jaccard’s coefficients ranged from 13.7 to 84.10% indicating a wide genetic base. Dendrogram was constructed by unweighted pair group method with arithmetic averages method. RAPD technology could be useful for identification of different accessions as well as assessing the genetic similarity among different genotypes of lettuce. The study reveals the limited genetic base and the needs to diversify using new sources from the germplasm.

Agrobacterium-Mediated Insect Resistance Gene (cry1Aa) Transfer Studies Pertaining to Antibiotic Sensitivity on Cultured Tissues of Broccoli

ABSTRACT Successful plant transformation requires an efficient regeneration protocol and a suitable selection method. A genetically engineered, disarmed Agrobacterium tumefaciens strain containing a binary vector pBin-1Aa with cry1Aa (insect resistance) and npt-II (neomycin phosphotransferase-II) genes was used for genetic transformation studies. Effects of antibiotics kanamycin and cefotaxime were studied to determine the aptness of kanamycin resistance as a selectable marker and for cefotaxime in controlling excessive bacterial growth during genetic transformation studies using cultured hypocotyl, cotyledon, leaf, and petiole tissues of Broccoli (Brassica oleracea L var. italica). Leaf and petiole explants exhibited decreased fresh weight as kanamycin concentration increased, resulting in full or partial inhibition of shoot regeneration. A significant or nonsignificant negative correlation occurred between kanamycin concentration and explant fresh weight over the time. Cefotaxime had little effect on regeneration potential. Growth of Agrobacterium cells was controlled at a concentration of 400 mg·L−1 cefotaxime in explants, and maximum putative transgenic shoot regeneration in explants of hypocotyls (44.88%) and cotyledons (36.29%) was obtained on MS (Murashige and Skoog)-selective shoot regeneration medium. Polymerase chain reaction analysis of genomic DNA using specifically designed primers detected the presence of the cryIAa and npt-II genes in kanamycin-resistant broccoli plantlets. Of five randomly selected putative transgenic shoots, three were positive for presence/integration of cryIAa and npt-II genes during T-DNA transfer and integration into the plant genome. Kanamycin and cefotaxime act as effective selective agents during genetic transformation pertaining to antibiotic sensitivity on cultured tissues of broccoli.

Morphogenic Potential of Different Explants of Broccoli (Brassica oleracea L. var. italica): Important “Nutrient-Rich” Vegetable, Using Thidiazuron

Broccoli is a high-valued nutritionally rich important cole vegetable crop. But its quality and quantity are largely affected by various pests, diseases, and environmental stresses. Traditional plant breeding technologies alone are not sufficient to control its massive losses because of no natural genetic variations present in its germplasm. Genetic manipulation can supplement the conventional agricultural practices for broccoli improvement. The objective of the present study was to evaluate the effectiveness of thidiazuron (TDZ) in in vitro high-frequency plant regeneration studies in broccoli using hypocotyl, cotyledon, petiole, and leaf explants. Highly efficient and reproducible regeneration protocol has been optimized in broccoli cv. Solan green head. Explants were cultured on Murashige-Skoog (MS) medium, containing different TDZ concentrations and combinations such as TDZ alone, TDZ + adenine, TDZ + NAA (naphthalene acetic acid), and TDZ + IAA (indole acetic acid). Total 36 combinations were used; the maximum shoot regeneration response was observed in hypocotyl explants (95.92%) on MS medium supplemented with 2.0 μM TDZ + 0.5 μM IAA followed by petiole explants (91.55%) on MS medium with 2.0 μM TDZ + 0.107 μM NAA. Leaf explants gave (89.25%) shoot regeneration response on MS medium containing 1.0 μM TDZ + 0.107 μM NAA and cotyledon explants producing multiple shoots (88.88%) on MS medium supplemented with 2.0 μM TDZ + 0.59 mM adenine. After completed shoot regeneration, high-frequency (100%) root regeneration response was observed on rooting medium containing 0.10 mg/l NAA. For hardening, in vitro regenerated plantlets were transferred to pots containing cocopeat and successfully acclimatized. Well developed in vitro plant regeneration protocol has been optimized in broccoli cv. Solan green head using potent cytokinin thidiazuron.

Genetic diversity and relationship assessment of tomato (Solanum lycopersicum L.) genotypes using molecular markers

Genetic variability and relationships among 21 genotypes of tomato were analyzed using PCR-based molecular markers. A total of 25 random decamer oligonucleotide primers were examined to generate RAPD profiles. Out of these, reproducible patterns were obtained with 15 primers. A total of 72 amplicons were obtained, out of which all were polymorphic and seven were unique bands. In case of SSR studies, however, nine primers were used and total number of amplicons obtained were 33 with three unique bands. The percentage of total polymorphism across genotypes was 100% in both RAPD and SSR studies. Genetic similarity matrix, based on Jaccards coefficients, ranged from 3.85% to 73.17% indicating a wide genetic base. Dendrogram was constructed by using unweighted pair group method with arithmetic averages method. The study showed the importance of RAPD and SSR markers for genetic analysis and indicated a considerable amount of genetic diversity among the different studied genotypes of tomato.