UNIVERSITI PUTRA MALAYSIA

Transcription

1 UNIVERSITI PUTRA MALAYSIA TOWARDS THE DEVELOPMENT OF A PROTOCOL FOR CRYOPRESERVATION OF EMBRYOS OF A RECALCITRANT SEED (ARTOCARPUS HETEROPHYLLUS LAM.) BASKARAN KRISHNAPILLAY FP

2 TOWARDS THE DEVELOPMENT OF A PROTOCOL FOR CRYOPRESERVATION OF EMBRYOS OF A RECALCITRANT SEED (ARTOCARPUS HETEROPHYLLUS LAM.) By BASKARAN KRISHN APILLA Y Thesis Submitted in Partial Fulfilment of the Requirements for the Degree of Doctor of Philosophy in the Faculty of Agriculture Universiti Pertanian Malaysia July 1989

3 DEDICATED to the memory of my late Father VARGHESE KRISHNAPILLAY and to my Mother whose patience and understanding has been a constant source of inspiration for me throughout this study

4 ACKNOWLEDGEMENT It pleases me to have this opportunity of expressing my sincere gratitude to Universiti Pertanian Malaysia and in particular the Faculty of Agriculture for enabling this study to be carried out. My sincere thanks are also due to the International Board for Plant Genetic Resources (IBPGR/FAO), Rome for financial support through their grant No. 85/114 (a) without which this study would not have been possible. I am extremely grateful and greatly indebted to my supervisor Professor Chin Hoong Fong for his dedicated efforts, stimulating discussions, active participation and constant encouragement throughout the planning and execution of this research. My sincere thanks also goes to my co-supervisor Dr Hor Yue Luan for his advice, constructive criticisms, constant encouragement and guidance throughout this study. I take this opportunity also to thank Dr Zaliha Christine Alang of the Biotechnology Department for allowing me the use of the Plant Tissue Culture Laboratory facilities for carrying out a major portion of this research and also for her constant assistance provided throughout this study. Thanks are also due to Dr Gan Yik Yuan for her help and guidance during protein separation studies using electrophoresis. iii

5 My sincere appreciation is extended to Mr Ho Oi Kuan for his technical assistance in the preparation of the electron micrographs; Mr How Peng Guan for technical assistance in the preparation of the histological slides; Capt. Yap Yen Kiew for the construction of the starchgel electrophoresis kit; Encik Abdul Ghani Hashim (Fakulti Sains dan Pengajian Alam Sekitar) for his professional photographic work; Encik Abdul Ghani Hashim (Unit Hydroponik) for the loan of his printer during the preparation of this manuscript; the Faculty of Forestry, U.P.M. for use of their misting and glasshouse facilities and Mr Ong Choon Hoe for his valuable friendship and assistance in the laboratory during the course of the study. Thanks are also due to the Director, Dr M M Guha of Agricultural Research and Advisory Bureau for allowing the use of his office facilities during the entire course of this study. Sincere thanks are due to Mr and Mrs Ramachandran for help provided for proof reading of the original drafts and in the preparation of the final manuscript. Lastly but not the least, I wish to thank all my family members especially Rajan, Richard, Sara and George Krishnapillay for being very understanding and for their constant encouragement and support throughout the course of this study. iv

6 TABLE OF CONTENTS ACKNOWLEDGEMENT LIST OF TABLES LIST OF PLATES LIST OF FIGURES LIST OF ABBREVIATIONS ABSTRACT ABSTRAK iii ix xiv xviii xix xx xxiii CHAPTERS INTRODUCTION Need for the Study Selection of Crop for the Study Objectives of the Study REVIEW OF LITERATURE Present State of Recalcitrant Seed Storage Characteristics of Recalcitrant Seeds Moisture Content of Recalcitrant Seeds Desiccation Sensitivity of Recalcitrant Seeds Chilling Sensitivity in Recalcitrant Seeds Potential of In Vitro Storage for Germplasm Conservation of Difficult Materials Cryopreservation of Embryos Chemical Dehydration and Toxicity Freezing Injury v

7 Mechanism of Natural Tolerance to Freezing Injury Cryoprotective Agents Use of Cryoprotective Compo\Ulds and Their Mixtures Thawing Recovery!Recovery Medium Summary MATERIALS AND METHODS Study Layout Seed Materials and Sampling Experimental Procedures Packaging and Storage Excision of Embryos Regulation of Embryo Moisture Glassware Cleaning for In Vitro Studies Chemicals Preparation of Stock Solutions and Culture Media Incubation of Cultures Preparation of Cryoprotectants Cooling and Thawing Procedures Preparation of Separation of Electrophoresis Starch Gel and Sample for Proteins by Starch Gel 47 Preparation of Sections for Light Microscopy 50 Preparation of Sections for Transmission Electron 51 Microscopy Photography 53 vi

8 3.4 Measurements and Observations 53 Moisture Content 53 Dry Matter Content and Shoot to Root Ratio 54 of In Vitro Grown Seedlings Conductivity of Embryo Leachate 57 Uptake of Oxygen During Respiration 57 Comparison of Some Metabolic Enzymes After 58 Electrophoretic Separation 3.5 Experiments 58 Selection of Optimal Stage of Fruit Maturity 58 for Excised Embryos (Experiments la - c) Selection of Embryos for Cryopreservation Studies 61 (Experiments 2a - b) Establishment of a Suitable Medium for the Growth 62 and Development of Excised Jackfruit Embryos (Experiments 3a - c) Dehydration Effects on Embryo Viability and 65 Storage (Experiments 4a - e) Use of Cryoprotectants (Experiments 5a - g) 69 Modification of Culture Medium for Improved 74 Growth and Planting Out of Cryopreserved Seedlings (Experiments 6a - b) Statistical Analysis RESULTS Selection of Optimum Stage of Maturity for Excised Embryos Selection of Embryos for Cryopreservation Studies Establishment of a Suitable Medium for the Growth and Development of Excised Jackfruit Embryos Dehydration Effects on Embryo Viability and Storage 103 vii

9 Use of Cryoprotectants Modification of Culture Medium for Improved Growth and Planting Out of the Cryopreserved Embryos DISCUSSION SUMMARY AND CONCLUSION BIBLIOGRAPHY APPENDIX A B C D VITAE Background of the Fruit Artocarpus heterophyllus Murashige and Skoog (1962) Inorganic Salts and Vitamiru; Preparation of Chemicals Anova Tables viii

10 LIST OF TABLES TABLE Viability Percentage of Embryos Excised from the Three Stages of Fruit Maturity, After Six Weeks in Culture Moisture Content and Viability Percentage of Excised Embryos from the Three Stages of Fruit Maturity After Desiccation Over a Period of Six Hours Categorizing Excised Embryos by Size, from a Random Sample of Seeds Taken from Mature Unripe Fruits Fresh Weight, Moisture Content and Coefficient of Variation of Moisture Content of Excised Embryos from the Various Selection Methods Mean Dry Matter Content of Plantlets Grown In Vitro on Media Containing NAA/Kinetin and With or Without Activated Charcoal Shoot to Root Ratio of Plantlets Grown In Vitro on Media Containing NAA/Kinetin and With or Without Activated Charcoal Mean Dry Matter Content of Plantlets Grown In Vitro on Media Containing N AA/BAP and With or Without Activated Charcoal Shoot to Root Ratio of Plantlets Grown In Vitro on Media Containing N AA/BAP and With or Without Activated Charcoal Mean Dry Matter Content of Plantlets Grown In Vitro on Media Containing N AA/2iP and With or Without Activated Charcoal Shoot to Root Ratio of Plantlets Grown In Vitro on Media Containing NAA/2iP and With or Without Activated Charcoal Moisture Content of Excised Embryos Obtained for the Various Methods of Drying In Vitro Survival Percentage of Excised Embryos After Various Methods and Periods of Drying ix

11 TABLE Moisture Content and Survival Percentage of Excised Embryos Desiccated Over a Moisture Range of 10-15% Physiological and Biochemical Changes in Excised Embryos Desiccated Over a Period of One to Six Hours Survival Percentage of Stored Excised Embryos (Desiccated to % and 12-13% Moisture Content Respectively) Cultured Weekly Over a Period of Four Weeks Survival Percentage of Excised Embryos Dried to Near Critical Moisture Level and Stored in Liquid Nitrogen Survival Percentage of Excised Embryos After Cryoprotecting in Various Combination of Cryoprotectants for Various Period of Time Survival Percentage of Excised Embryos Cryoprotected for Six Hours Followed by Storage Treatments and Direct Placement in Liquid Nitrogen Survival Percentage of Excised Embryos Cryoprotected for Twelve Hours Followed by Storage Treatments and Direct Placement in Liquid Nitrogen Moisture Content in Excised Embryos After Cryoprotection and Desiccation for One, Two and Three Hours Survival Percentage of Cryopreserved Embryos After Cryoprotection and Desiccation for One, Two and Three Hours Survival Percentage of Excised Embryos After Twelve Hours of Cryoprotection Followed by Partial Desiccation for One and Two Hours and Two Methods of Cooling Survival Percentage of Cryoprotected Embryos Partially Desiccated and Subjected to Slow Freezing Protocol x

12 TABLE Assessment of Viability of Cryopreserved Embryos Stored in Liquid Nitrogen Over a Period of One Month Using the Slow Freezing Protocol Mean Dry Matter Content and Shoot to Root Ratio of Control and Cryopreserved Embryos Cultured on Two Different Media Survival Percentage of Control and Cryopreserved Seedlings in Polybags After Eight Weeks of Planting in the Nursery ANOVA Table of Viability Percentage of Excised Embryos from Three Stages of Fruit Maturity After Six Weeks in Culture Mean Square Values for Minimum, Maximum, Mean and Range of Fresh Weights of Excised Embryos from the Various Selection Methods Mean Square Values for Minimum, Maximum, Mean, Range and Coefficient of Variation in Moisture Content of Excised Embryos from the Various Selection Methods AN OV A Table of Mean Dry Matter Content of Plantlets Grown In Vitro on Media Containing NAA/Kinetin and With or Without Activated Charcoal ANOVA Table of Shoot to Root Ratio of Plantlets Grown In Vitro on Media Containing N AA/Kinetin and With or Without Activated Charcoal ANOVA Table of Mean Dry Matter Content of Plantlets Grown In Vitro on Media Containing NAA/BAP and With or Without Activated Charcoal ANOVA Table of Shoot to Root Ratio of Plantlets Grown In Vitro on Media Containing NAA/BAP and With or Without Activated Charcoal ANOVA Table of Mean Dry Matter Content of Plantlets Grown In Vitro on Media Containing NAA/2iP and With or Without Activated Charcoal AN OV A Table of Shoot to Root Ratio of Plantlets Grown In Vitro on Media Containing N AA/2iP and With or Without Activated Charcoal 218 xi

13 TABLE ANOVA Table of Moisture Content and Survival Percentage of Excised Embryos Desiccated Over a Moisture Range of 10-15% Mean Square Values of Moisture Content, In Vitro Survival Percentage, Leachate Conductivity, Tetrazolium Test and Respiration Rate of Excised Embryos Desiccated for a Period of One to Six Hours Mean Square Values of Survival Percentage of Stored Excised Embryos (Desiccated to 14-15% Moisture Content), Cultured Weekly Over a Period of Four Weeks Mean Square Values of Survival Percentage of Stored Excised Embryos (Desiccated to 12-13% Moisture Content), Cultured Weekly Over a Period of Four Weeks AN OV A Table of Survival Percentage of Excised Embryos Dried to Near Critical Moisture Level and Stored in Liquid Nitrogen ANOVA Table of Survival Percentage of Embryos After Cryoprotecting in Combination of Cryoprotectants for Period of Time Excised Various Various ANOVA Table of Survival Percentage of Excised Embryos Cryoprotected for Six Hours Followed by Storage Treatments and Direct Placement in Liquid Nitrogen AN OV A Table of Survival Percentage of Excised Embryos Cryoprotected for Twelve Hours Followed by Storage Treatments and Direct Placement in Liquid Nitrogen ANOVA Table of Survival Percentage of Cryopreserved Embryos After Cryoprotection and Desiccation for One, Two and Three Hours AN OV A Table of Survival Percentage of Excised Embryos After Twelve Hours of Cryoprotection Followed by Partial Desiccation for One and Two Hours and Two Methods of Cooling xii

14 TABLE ANOVA Table of Survival Percentage of Cryoprotected Embryos Partially Desiccated and Subjected to Slow Freezing Protocol AN OV A Table of Assessment of Cryopreserved Embryos Stored in Liquid Nitrogen Over a Period of One Month Using the Slow Freezing Protocol Mean Square Values for Mean Dry Matter Content and Shoot to Root Ratio of Control and Cryopreserved Embryos Cultured on Two Different Media AN OV A Table of Survival Percentage of Control and Cryopreserved Seedlings in Polybags After Eight Weeks of Planting in the Nursery xiii

15 LIST OF PLATES PLATE 1 A Jackfruit of Variety NS Excision of the Sharp Scalpel Embryo of Jackfruit Using a The Selected Three Stages of Fruit Along With Their Seeds (a) Fully Ripe (b) Ripe Fruit (c) Mature Unripe Fruit Developing Plantlets from Embryos Excised from Fruits at the Three Stages of Maturity After Six Weeks in Culture (a) Fully Ripe Fruit (b) Ripe Fruit (c) Mature Unripe Fruit (Culture tube diam. 25 mm) Comparison of Four Metabolic Enzymes in the Embryos from Fruits at the Three Stages of Maturity (a) Fully Ripe Fruits (b) Ripe Fruits (c) Mature Unripe Fruits Variability in Embryo Size Excised from a Random Sample of Seeds Close up of Selected Embryo Measuring mm Used in the Study Plantlet Development in Medium Containing 0.1 mg/l each of NAA and Kinetin in the Absence of Charcoal After Eight Weeks of Growth (Culture tube diam. 25 mm) Plantlet Development in Medium Containing 1.0 mg/l each of NAA and Kinetin in the Presence of Charcoal, After Eight Weeks of Growth (Culture tube diam. 25 mm) Plantlet Development in Medium Containing 0.1 mg/l each of NAA and BAP in the Absence of Charcoal, After Eight Weeks of Growth (Culture tube diam. 25 mm) Plantlet Development in Medium Containing 1.0 mg/l each of N AA and BAP in the Presence of Charcoal, After Eight Weeks of Growth (Culture tube diam. 25 mm) xiv

16 PLATE Plantlet Development in Medium Containing 0.1 mg/! each of NAA and 2iP in the Absence of Charcoal, After Eight Weeks of Growth (Culture tube diam. 25 mm) Plantlet Development in Medium Containing 1.0 mg/! each of N AA and 2iP in the Presence of Charcoal, After Eight Weeks of Growth (Culture tube diam. 25 mm) Comparison of Root Development in Media With and Without Charcoal Viability of Embryos Dried Over a Period of Six Hours by Ambient Drying Method (Culture tube diam. 20 mm) Viability of Embryos Dried Over a Period of Six Hours by Air-Condition Drying Method (Culture tube diam. 20 mm) Viability of Embryos Dried Over a Period of Six Hours by Laminar Air Flow Drying Method (Culture tube diam. 20 mm) Viability of Embryos Dried Over a Period of Six Hours by Silica-Gel Drying Method (Culture tube diam. 20 mm) Stunted Growth of Embryos Dried to Moisture Content of 9-10% Embryo Growth and Development After Drying to Six Moisture Levels in a Laminar Air Flow Cabinet (a) 14-15% (b) 13-14% (c) 12-13% (d) 11-12% (e) % (f) 9-10% Cellular Structure of Radicle Tip and Cortex from Excised Embryos of Fresh Jackfruit Seeds (0 Hours of Drying) (a) Radicle Tip (x 200) (b) Cortex Tissue of Radicle (x 400) Cellular Structure of Radicle Tip and Cortex from Excised Embryos Subjected to One Hour of Drying in a Laminar Air Flow Cabinet (a) Radicle Tip (x 400) (b) Cortex Tissue of Radicle (x 400) Cellular Structure of Radicle Tip and Cortex from Excised Embryos Subjected to Two Hours of Drying in a Laminar Air Flow Cabinet (a) Radicle Tip (x 400) (b) Cortex Tissue of Radicle (x 400) xv

17 PLATE Cellular Structure of Radicle Tip and Cortex from Excised Embryos Subjected to Three Hours of Drying in a Laminar Air Flow Cabinet (a) Radicle Tip (x 400) (b) Cortex Tissue of Radicle (x 400) Cellular Structure of Radicle Tip and Cortex from Excised Embryos Subjected to Four Hours of Drying in a Laminar Air Flow Cabinet (a) Radicle Tip (x 400) (b) Cortex Tissue of Radicle (x 400) Cellular Structure of Radicle Tip and Cortex from Excised Embryos Subjected to Five Hours of Drying in a Laminar Air Flow Cabinet (a) Radicle Tip (x 400) (b) Cortex Tissue of Radicle (x 400) Cellular Structure of Radicle Tip and Cortex from Excised Embryos Subjected to Six Hours of Drying in a Laminar Air Flow Cabinet (a) Radicle Tip (x 400) (b) Cortex Tissue of Radicle (x 400) Embryos at 14-15% Moisture Showing Survival After Storage in Liquid Nitrogen for Twelve Hours (Culture tube diam. 25 mm) Embryos at 13-14% Moisture Showing Survival After Storage in Liquid Nitrogen for Twelve Hours (Culture tube diam. 25 mm) Embryos at 12-13% Moisture Showing Survival After Storage in Liquid Nitrogen for Twelve Hours (Culture tube diam. 25 mm) Embryos Showing Either Shoot or Root Development After Cryoprotection Followed by Rapid Freezing After Four Weeks in Culture Embryos Showing Normal Development of Shoot and Root After Cryoprotection Followed by Slow Freezing After Four Weeks in Culture Ultrastructure of Radicle Cells from Fresh Jackfruit Embryo Ultrastructure of Radicle Cells from Jackfruit Embryos Cryopreserved After Desiccating to % Moisture Content 141 xvi

18 PLATE 35 Ultrastructure of Radicle Cells from Jackfruit Embryos Cryopreserved After Desiccating to 12-13% Moisture Content Ultrastructure of Radicle Cells from Jackfruit Embryos Cryopreserved After Desiccating to 9-1 0% Moisture Content Ultrastructure of Radicle Cells from Jackfruit Embryos Cryopreserved Directly in Liquid Nitrogen After Twelve Hours of Cryoprotection and Partial Desiccation to 29-33% Moisture Content Ultrastructure of Radicle Cells from Jackfruit Embryos Cryopreserved After Twelve Hours of Cryoprotection Followed by Partial Desiccation to 29-33% Moisture Content and Slow Cooling to -40 C Control Embryos Developing on Initially Established and Enriched Media (a) Initially Established Medium (b) Enriched Medium Cryopreserved Embryos Developing on Initially Established and Enriched Media (a) Initially Established Medium (b) Enriched Medium Plantlets in Glasshouse After Removal from the Misting Chamber Comparison Between Control and Cryopreserved Plantlets After Five Weeks in a Misting Chamber (a) Control Plantlet (b) Cryopreserved Plantlet Fresh Seeds and Seeds Stored for Three Days (a) Fresh Seeds (b) Seeds Stored for Three Days Showing Signs of Germination Jackfruit Trees in a Small Scale Plantation Close up of Jackfruits Developing on the Tree A Section Through a Jackfruit Showing the Fruit (f), Seed (s) and Embryo (e) 200 xvii

19 LIST OF FIGURES FIGURE 1 Graph Showing Rate of Cooling for the Alcohol Bath (Julabo - Model F40-HC) 48 xviii

20 LIST OF ABBREVIATIONS The following abbreviations were used in the text : NAA BAP 2iP LSD DNMRT RH EtOH TBA w/w v/v min C mg g I J.Ll Kg/cm 2 ).18 gil mg/l MS % DMSO Kg rpm GA 3 olj-napthalene acetic acid Benzylam inopurine 2-isopentyl adenine Least Significant Difference Duncan's New Multiple Range Test Relative Humidity Ethyl alcohol Tertiary Butyl Alcohol weight by weight volume by volume minutes Degree centigrade milligram gram litre microlitre Kilogram per square centimetre Microsemens grams per litre miligram per litre Murashige and Skoog Medium formulation percentage Dimethysulf oxide Kilogram revolution per minute Gibberellic acid xix

21 ABSTRACT Abstract of the thesis submitted to the Senate of Universiti Pertanian Malaysia in partial fulfilment of the requirements for the Degree of Doctor of Philosophy. TOWARDS THE DEVELOPMENT OF A PROTOCOL FOR CRYOPRESERVA110N OF EMBRYOS OF A RECALCITRANT SEED (ARTOCARPUS HETEROPHYLLUS LAM.) by BASKARAN KRISHNAPILLAY July, 1989 Supervisor Co-Supervisor Faculty Professor Chin Hoong Fong Dr Hor Yue Luan Agriculture Long term storage of recalcitrant seeds by conventional storage methods have by far been unsuccessful. In this study the excised embryos of seeds of jackfruit (Artocarpus heterophyllus). a recalcitrant species has been attempted at for long term preservation using cryogenic means. Stage of fruit maturity was critical in the study where embryos excised from mature unripe fruits were found to be the most appropriate. xx

22 Selection of uniform sized embryos ( mm in length) minimised variability in embryo fresh weight and moisture content to a considerable extent. A modified Murashige and Skoog medium containing 1.0 mgll each of NAA and BAP, 2 gil of activated charcoal and 170 mgll of NaH 2 P0 4.H 2 0 was found to be a suitable medium for development and growth of the excised jackfruit embryos. The safe critical moisture limit for excised jackfruit embryos was found to be around %. Storage of embryos dried to this moisture limit by conventional storage methods with a view to long term storage was unsuccessful. The maximum storage period achieved was only one month. Attempts at direct storage in liquid nitrogen after desiccation to near critical moisture limit was also found to be unsuccessful. Embryos cryoprotected with 10% DMSO + 0.5% proline for 12 hours followed by a partial desiccation to moisture contents of 29-33% and slowly frozen to -40 C at approximately 1 C/min before plunging into liquid nitrogen survived cryopreservation and gave approximately 60% normal recovery. No significant decline in the viability percentage was observed in those embryos stored by this method for a period of one month. For normal recovery growth of embryos after cryopreservation, addition of other growth promoting substances namely GA 3 ' glutamine, argenine, asparagine and adenine sulphate to the initially established medium was found to be beneficial. xxi

23 Only 50% of the well developed plantlets transferred to polythene bags were able to survive the transplanting shock and resume normal growth in the glasshouse. The plantlets required a hardening period of five weeks in a high humidity chamber before they were able to withstand the ambient conditions in the glasshouse. xxii

24 ABSTRAK Abstrak tesis yang dikemukakan kepada Senat Universiti Pertanian Malaysia bagi memenuhi sebahagian daripada syarat-syarat untuk memperolehi Ijazah Doktor Falsafah. TOWARDS THE DEVELOPMENT OF A PROTOCOL FOR CRYOPRESERVATION OF EMBRYOS OF A RECALCITRANT SEED (ARTOCARPUS HETEROPHYLLUS LAM.) oleh BASKARAN KRISHN APILLAY July, 1989 Penyelia Penolong Penyelia Fakulti Professor Chin Hoong ong Dr Hor Yue Luan Pertanian Penyimpanan biji benih tempoh panjang melalui kaedah penyimpanan biasa, belum berjaya setakat ini. Dalam kajian ini, embrio-embrio yang telah diasingkan dari biji benih nangka (Artocarpus heterophyllus), sejenis spesies rekalsitran, telah dikaji sebagai bahagian tumbuhan untuk penyimpanan tempoh panjang melalui kaedah kriogenik. xxiii

25 Peringkat kematangan buah didapati sangat penting. Embrio dari biji benih dalam buah yang matang tetapi belum masak, didapati yang sesuai sekali. Pemilihan embrio yang saiznya seragam ( mm panjangnya) didapati mengurangkan variabiliti dalam berat segar dan kandungan kelembapan embrio. Medium Murashige dan Skoog diubahsuai, yang mengandungi 1.0 mgll N AA dan BAP masing-masing, 2 gil arang diaktifkan dan 170 mgll NaH 2 P0 4.H 2 0 didapati sesuai untuk perkembangan dan tumbesaran embrio nangka. Untuk embrio nangka, had kritikal kelembapan didapati lebih kurang %. Ujian untuk menyimpan embrio yang dikeringkan kepada kelembapan ini, melalui kaedah biasa tidak berjaya. Tempoh penyimpanan maksimum yang dicapai hanyalah selama sebulan sahaja. Percubaan untuk menyimpan embrio secara terus dalam nitrogen cecair selepas dikeringkan sehingga hampir had kritikal, juga didapati tidak sesuai. Embrio yang dirawat dengan DMSO 10% + 0.5% prolin selama 12 jam diikuti oleh separuh kekeringan sehingga 29-33% kelembapan dan dibekukan dengan perlahan pada kadar l C/min sehingga -40 C sebelum diletakkan kedalam nitrogen cecair, didapati terus hidup dan menunjukkan kepulihan lebih kurang 60%. Tiada penurunan yang bererti dalam peratus viabiliti bagi embrio yang disimpan melalui kaedah ini selama sebulan. Untuk memperolehi tumbesaran normal bagi em brio selepas krioawetan, penambahan bahan penggalakan tumbesaran (iaitu GA 3, xxiv