Research Areas

Genetic Resources 2009-2013

Researcher: Alfredo Augusto Cunha Alves

Objectives of Labex Genetic Resources

To increase the activities of germplasm exchange between Brazil and USA; to expand partnerships between Embrapa and US researchers on innovative and strategic research; and to perform research activities related to cassava germplasm long-term conservation.

Project(s)

The main areas of the Labex Genetic Resources research program, led by Dr. Alfredo Alves, are related to ‘Biotechnological procedures for long-term preservation and evaluation of the diversity of plant genetic resources’, where studies on in vitro micropropagation and cryopreservation techniques are the main focus. The main crop for these studies has been cassava, with some preliminary studies with sugarcane. In 2012, new crops were included, such as Eucalyptus, Annanas, and Citrus with most of the activities developed by a group of Brazilian visiting scientists, who started the research work in 2011, at ARS-Fort Collins (LABEX cluster).

Development of cryopreservation system for multiple Manihot species

Research team: Alfredo Alves – Embrapa/LABEX, Fort Collins CO; Leonardo Rodrigues – Federal University of Lavras-Brazil and NCGRP/ARS, USA; Ergun Kaya – Gebze Institute of Technology-Kocaeli-Turkey and NCGRP/ARS, USA; David Ellis – NCGRP/ARS, Fort Collins CO; and Maria Jenderek – NCGRP/ARS, Fort Collins CO.

Objectives: To evaluate cryogenic procedures for cassava cryopreservation; and to develop a cryopreservation system that can be used for many species and accessions of Manihot (cultivated and wild relatives).

Approach: Evaluation of the Droplet-Vitrification procedure was done on 2-3 months old in vitro plants of at least 4 Manihot species obtained via micropropagation at NCGRP. Excision of uni-nodal stem segments (~1cm) and cultivation on CIAT 12A3 medium, in Petri dishes (90 x 25 mm). After 14-21 days, apical shoot tips (1.0-1.5 mm) were excised and inoculated onto a solid Pre-Culture medium (MS supplemented with 0.3M sucrose) in small Petri dishes (55 x 15 mm) for 16-24 hours in a growth chamber under indirect light. Shoot tips (10-15) were placed in Loading Solution (LS) consisting of MS supplemented with 2M glycerol and 0.4M sucrose for 20 minutes. LS was removed and shoot tips was placed into a small drop (5 μL) of Plant Vitrification Solution (PVS2) on a small (~5 x 15mm) aluminum foil strip (5 drops/strip). Different exposure times of PVS2 have been tested (15, 30, 45, 60 minutes). These two solutions (LS + PVS2) are the main steps for the vitrification process. A group of vitrified shoot tips was cultured in regeneration medium (PVS2-Control) and another group plunged in liquid nitrogen-LN (PVS2-LN). After 24h, cryopreserved meristems were removed from LN, quickly placed in 1.2 M sucrose solution for 20 min., cultured in a regeneration medium and maintained in a growth chamber along with the PVS2-Control plates. Evaluation of growth and regeneration rates were made weekly.

Results: After 5-6 weeks, treatments with PVS2-Controls showed no toxicity and some shoot tips, exposed to LN, presented callus formation with or without leaf shoots, showing good regeneration after cryo treatment. In both cultivated species and two wild species (M. chlorostica and M. flabellifolia), there was full regeneration of plants with different rates after LN, suggesting that the procedure appears promising for these species and to be tested in other species. The recent regeneration data are being analyzed.

Multiplication, expansion and conservation of Manihot species collection at NCGRP/ARS

Research team: Alfredo Alves – Embrapa/LABEX, Fort Collins CO; Leonardo Rodrigues – Federal University of Lavras-Brazil and NCGRP/ARS, USA; David Ellis – NCGRP/ARS, Fort Collins CO; and Maria Jenderek – NCGRP/ARS, Fort Collins CO.

Objectives: To conserve a collection of Manihot species in the NCGRP under suitable conditions; to expand the NCGRP collection through introductions of new accessions and species of Manihot; and to transfer the in vitro collection of Manihot species from NCGRP to Embrapa Cassava & Fruits.

Approach: Multiplication of the in vitro Manihot collection every 3-6 months and storage of the wild cassava seeds collection at low temperature (5°C). Expansion of in vitro collection through introductions of new species and accessions via production and micropropagation of seedlings obtained from sexual seeds. In vitro micropropagation of seedlings has been successfully achieved by cleaning the apical and lateral meristems with 50% isopropyl alcohol for 1 minute, followed by 3% sodium hypochlorite for 2 min. After cleaning the explants are cultivated in CIAT 12A3 medium.

Results: A total of 88 accessions of 25 species of Manihot (including commercial, M. esculenta, with six varieties) has been conserved in vitro, which represents the material transferred from CIAT (46 accession) and new introductions from seedlings produced in greenhouse and micropropagated in vitro (42 accessions). Each access is maintained in at least three magenta bottles (G7) with 5 plants per vial. The list of the collection is in Table 01. Collection of seeds from 13 species of Manihot has been kept in cold storage (5oC).

Optimize methodologies to establish NPGS sugarcane accessions under in vitro tissue culture

Research team: Alfredo Alves – Embrapa/LABEX, Fort Collins CO; David Ellis – NCGRP/ARS, Fort Collins CO; Ergun Kaya – Gebze Institute of Technology-Kocaeli-Turkey and NCGRP/ARS, USA; Leonardo Rodrigues – Federal University of Lavras-Brazil and NCGRP/ARS, USA; Maria Jenderek – NCGRP/ARS, Fort Collins CO; Thomas Ayala-Silva – Subtropical Horticulture Research Station-SHRS/ARS, Miami FL; and Clarissa Maroon-Lango – Animal and Plant Health Inspection Service-APHIS/USDA, Beltsville MD.

Objectives: To develop protocols for in vitro tissue culture and cryopreservation of NPGS sugarcane accessions; and to establish NPGS accessions of sugarcane in tissue culture and free from diseases suitable for transfering to Brazil via NPGS.

Approach: Accessions of sugarcane maintained in the field at Subtropical Horticulture Research Station-SHRS, Miami, FL and from Animal and Plant Health Inspection Service-APHIS, Beltsville, MD have been sent to NCGRP. The shipment of apical shoots from APHIS has continued to expand the number of accessions to be multiplied or regenerated at NCGRP. So far, the accessions selected by the sugarcane curator, represent a wide genetic diversity of frequently requested and readily available sugarcane accessions from the NPGS sugarcane collection. As needed, additional research in cryopreservation will be done using one or a combination of vitrification, encapsulation, dehydration and/or slow cooling techniques.

Results: The follow up and expansion of this project will count on Embrapa’s collaboration on sending a researcher to NCGRP to perform micropropagation and cryopreservation research and accelerate the introduction of materials to Brazil. In vitro production of 32 accessions of sugarcane, free of viruses and diseases, which were multiplied successfully and are being maintained at NCGRP.

Phenotyping of cassava to identify QTLs for drought tolerance

In addition to research work in the US, the Labex scientist also has activities underway in Brazil, coordinating this international project, funded by the 'Generation Challenge Program-GCP', involving the direct collaboration of researchers from Embrapa Cassava & Fruits, Embrapa Semi-Arid, and Cornell University.

Research team: Alfredo Alves – Embrapa/LABEX, Fort Collins CO; Maurício Coelho – Embrapa Mandioca e Fruticultura, Cruz das Almas BA; Vanderlei Santos – Embrapa Mandioca e Fruticultura, Cruz das Almas BA; Alineaurea Silva – Embrapa Semi-Árido, Petrolina PE; and Tim Setter – Cornell University, Ithaca, NY

Objective: Phenotyping in field conditions, a segregating population of cassava for drought tolerance.

Approach: A total of 128 individuals from a F1 segregating population generated with the cross COL 1734 x VEN 77 were evauated. A field trial was planted in 2010, in Petrolina-PE, a semi-arid region of Northeast Brazil with the following treatments: (1) Well watered (WW), full irrigation and (2) Water stress (WS) with irrigation until 3 months after planting (MAP). Plants were harvested at 4 MAP (one month after stopping irrigation) and 12 MAP, in 2011. Parameters that were evaluated at 4 MAP: (1) Plant growth and leaf retention; (2) Leaf conductance and leaf temperature; (3) carbohydrate and ABA in stems and leaves; (4) Weight, length and diameter of tuber roots (1 plant per plot) to evaluate initial root tuberization; and (5) Green mites damage evaluation. Parameters that were evaluated at 12 MAP: (1) Plant survival; (2) Branching habit; (3) Root shape and color; (4) Root tuber and shoots weight; and (5) Dry matter content (%DM) and harvest index (HI). The soil moisture in 5 different depths (10, 20, 30, 40, 50 cm) were evaluated monthly from Jul/10 (planting) until Mar/11 (7 MAP). Data analysis in 2012.

Results: During the 12 months of experimental period the total rainfall was only 469 mm, with a prolonged water deficit in the first 5 months. The irrigation helped to keep the soil moisture in higher levels in relation to rainfed condition. A total of 122 genotypes (out of 128 planted) were evaluated at 4 MAP and 12 MAP and a great diversity was observed in most parameters assessed both in well-watered (WW) and water-stressed (WS) conditions. The water stress caused a significant reduction in root yield of the population, but some individuals showed good performance under WS, under WW, and in both WS and WW conditions. Similar behavior also occurred with the yield of shoots. Some genotypes had a good performance of dry matter production and harvest index in response to water deficit . Osmolyte concentrations in stems, exhibited sufficient range among genotypes and precision that it may be feasible to use this as a phenotyping trait. For leaves, ABA had significant genotypic effects, providing encouragement that it can be useful for phenotyping. Among the carbohydrates, leaf sucrose is the most promising trait for phenotyping; as a compatible solute, it may relate to genotypic differences to stress tolerance.